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53898
ER Homeostasis Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

ER Homeostasis Antibody Sampler Kit #53898

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ER Homeostasis Antibody Sampler Kit: Image 1
Western blot analysis of extracts from 293 and HeLa cells, untreated (-) or tunicamycin-treated (2 μg/ml, 8 hr; +), using ATF-4 (D4B8) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
ER Homeostasis Antibody Sampler Kit: Image 2
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ASNS, a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
ER Homeostasis Antibody Sampler Kit: Image 3
Western blot analysis of extracts from various cell lines using BiP (C50B12) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 4
Western blot analysis of extracts from various cell types using PERK (C33E10) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 5
Western blot analysis of extracts from various cell lines, using IRE1α (14C10) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 6
Western blot analysis of extracts from C2C12 cells, untreated or thapsigargin-treated, using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb (upper) or eIF2α Antibody #9722 (lower).
ER Homeostasis Antibody Sampler Kit: Image 7
Western blot analysis of extracts from 293T cells untreated (-) or treated with Tunicamycin #12819 (2 μg/ml, 8 hr; +), using XBP-1s (E9V3E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
ER Homeostasis Antibody Sampler Kit: Image 8
Western blot analysis of extracts from 293T cells, untreated (-) or treated with tunicamycin (1 mM; 1hr; +), using ATF-6 (D4Z8V) Rabbit mAb. 
ER Homeostasis Antibody Sampler Kit: Image 9
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
ER Homeostasis Antibody Sampler Kit: Image 10
Western blot analysis of extracts from various cell lines using CCPG1 (E3C5G) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Absence of signal in Jurkat cells is predicted by RNAseq and confirms the specificity of the antibody.
ER Homeostasis Antibody Sampler Kit: Image 11
Western blot analysis of extracts from SNU-475 cells, untreated (-) or treated with Tunicamycin #12819 (5 μg/mL, 24 hr; +), using CCPG1 (E3C5G) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
ER Homeostasis Antibody Sampler Kit: Image 12
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human CCPG1 protein (hCCPG1-Myc/DDK; +), using CCPG1 (E3C5G) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
ER Homeostasis Antibody Sampler Kit: Image 13
Immunoprecipitation of CCPG1 protein from Colo 205 extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CCPG1 (E3C5G) Rabbit mAb. Western blot analysis was performed using CCPG1 (E3C5G) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
ER Homeostasis Antibody Sampler Kit: Image 14
Western blot analysis of extracts from various cell lines using FAM134B (E8Y9R) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal for FAM134B in Hep G2 cells is consistent with RNAseq data and confirms the specificity of the antibody for FAM134B.
ER Homeostasis Antibody Sampler Kit: Image 15
Immunoprecipitation of ATF-4 from extracts of 293 cells, treated with tunicamycin (2 μg/ml, 8 hr), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ATF-4 (D4B8) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ATF-4 (D4B8) Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.
ER Homeostasis Antibody Sampler Kit: Image 16
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 7 (upper), including ASNS (lower), a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
ER Homeostasis Antibody Sampler Kit: Image 17
Immunohistochemical analysis of paraffin-embedded human glioblastoma using BiP (C50B12) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 18
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, untreated (left) or λ phosphatase-treated (right), using Phopsho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 19
Western blot analysis of extracts from C2C12 cells untreated (-) or treated with Tunicamycin #12819 (2 μg/ml, 8 hr; +) and INS-1 cells untreated (-) or treated with Tunicamycin #12819 (5 μg/ml, 24 hr; +), using XBP-1s (E9V3E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
ER Homeostasis Antibody Sampler Kit: Image 20
Immunoprecipitation of ATF-6 from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ATF-6 (D4Z8V) Rabbit mAb. Western blot analysis was performed using ATF-6 (D4Z8V) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 21
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human FAM134B protein (hFAM134B-Myc/DDK; +), using FAM134B (E8Y9R) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
ER Homeostasis Antibody Sampler Kit: Image 22
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or tunicamycin-treated (2 μg/ml, 8 hr; right), using ATF-4 (D4B8) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
ER Homeostasis Antibody Sampler Kit: Image 23
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and either ATF-4 (D4B8) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human TRB3 exon 1 primer, human ASNS exon 1 primer and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
ER Homeostasis Antibody Sampler Kit: Image 24
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using BiP (C50B12) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 25
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 26
Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or treated with Tunicamycin #12819 (2 µg/ml, 8 hr; right), using XBP-1s (E9V3E) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
ER Homeostasis Antibody Sampler Kit: Image 27
Immunoprecipitation of FAM134B protein from SCLC-21H cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is FAM134B (E8Y9R) Rabbit mAb. Western blot analysis was performed using FAM134B (E8Y9R) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
ER Homeostasis Antibody Sampler Kit: Image 28
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across Ddit3/CHOP, a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
ER Homeostasis Antibody Sampler Kit: Image 29
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using BiP (C50B12) Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 30
Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
ER Homeostasis Antibody Sampler Kit: Image 31
Flow cytometric analysis of C2C12 cells, untreated (blue, low) or treated with tunicamycin (2 μg/mL, 8 hours; green, positive) using XBP-1s (E9V3E) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
ER Homeostasis Antibody Sampler Kit: Image 32
Confocal immunofluorescent analysis of TT cells (left, positive) and Hep G2 cells (right, negative) using FAM134B (E8Y9R) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
ER Homeostasis Antibody Sampler Kit: Image 33
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 10 (upper), including Ddit3/CHOP (lower), a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
ER Homeostasis Antibody Sampler Kit: Image 34
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using BiP (C50B12) Rabbit mAb in the presence of control peptide (left) or BiP Blocking Peptide #1084 (right).
ER Homeostasis Antibody Sampler Kit: Image 35
Chromatin immunoprecipitations were performed with cross-linked chromatin from C2C12 cells differentiated with 10% horse serum for 7 days then treated with tunicamycin (2 ug/ml) for 7 hours and either XBP-1s (E9V3E) Rabbit mAb #2392 or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human DNAJB9 Exon 1 Primers #79879, mouse EIF2AK3 promoter primers and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
ER Homeostasis Antibody Sampler Kit: Image 36
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight, and ATF-4 (D4B8) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse ATF-3 Intron 1 Primers #13059, mouse CHOP promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
ER Homeostasis Antibody Sampler Kit: Image 37
Flow cytometric analysis of A204 cells using BiP (C50B12) Rabbit mAb (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
To Purchase # 53898
製品# サイズ 数量 価格 在庫
53898T
1 Kit  (9 x 20 microliters)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
FAM134B (E8Y9R) Rabbit mAb 83414 20 µl
  • WB
  • IP
  • IF
H M R 70 Rabbit IgG
CCPG1 (E3C5G) Rabbit mAb 80158 20 µl
  • WB
  • IP
H 105-120 Rabbit IgG
XBP-1s (E9V3E) Rabbit mAb 40435 20 µl
  • WB
  • IF
  • F
  • ChIP
H M R 60 (human), 55 (mouse/rat) Rabbit IgG
ATF-6 (D4Z8V) Rabbit mAb 65880 20 µl
  • WB
  • IP
H M 90-100 Rabbit IgG
Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb 3398 20 µl
  • WB
  • IP
  • IHC
H M R Mk Dm 38 Rabbit IgG
PERK (C33E10) Rabbit mAb 3192 20 µl
  • WB
H M R Mk 140 Rabbit IgG
ATF-4 (D4B8) Rabbit mAb 11815 20 µl
  • WB
  • IP
  • IF
  • ChIP
  • C&R
H M R 49 Rabbit IgG
IRE1α (14C10) Rabbit mAb 3294 20 µl
  • WB
  • IP
H M R 130 Rabbit IgG
BiP (C50B12) Rabbit mAb 3177 20 µl
  • WB
  • IHC
  • F
H M 78 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The ER Homeostasis Antibody Sampler Kit provides an economical means of detecting proteins involved in ER homeostasis by regulating ER stress and ER-phagy. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the ER Homeostasis Antibody Sampler Kit detects endogenous levels of its target protein. XBP-1s (E9V3E) Rabbit mAb detects bands at 78 kDa and 99 kDa of unknown identity.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Pro327 of human FAM134B protein, Ala361 of mouse XBP-1s protein, Ala246 of human PERK protein, His963 of human IRE1α protein, Gly584 of human BiP protein, residues near the carboxy terminus of human ATF-6 and ATF-4 protein, and a synthetic phosphopeptide corresponding to residues surrounding Ser51 of human eIF2α protein.

Background

The endoplasmic reticulum (ER) is a large organelle extending from the nuclear envelope to the plasma membrane with diversity in structure and function (1,2). It functions in calcium storage, lipid and steroid synthesis, and protein folding, processing, and transport. ER structure and function is regulated in a dynamic fashion adapting to situations of ER stress and organelle damage (1,2). When demands on protein processing exceeds capabilities, cells trigger an adaptive mechanism called the unfolded protein response (UPR) which is largely controlled by the activities of three pathways: PERK, IRE1α, and ATF-6 (1). The ER chaperone protein BiP is recruited to unfolded proteins in the ER lumen and its dissociation from PERK, IRE1α, and ATF-6 leads to their activation. PERK is a kinase on the ER membrane that couples ER stress to changes in translation. PERK activation during ER stress leads to phosphorylation of eIF2α, repressing most translation but selectively inducing some targets such as ATF-4, a transcription factor that regulates targets in the recovery of the stress response. IRE1α is an ER protein with endoribonuclease activity that is activated during ER stress and converts XBP-1 from an unspliced XBP-1μ isoform to a spliced XBP-1s isoform functioning as a transcription factor regulating stress response genes. Lastly, during ER stress, ATF-6 is cleaved liberating a mature transcription factor controlling stress response genes.
Subsequent to ER expansion triggered by the UPR, cells may trigger a process of ER-phagy, the degradation of ER fragments through autophagy (3). Autophagy is a process for the bulk degradation of cytoplasmic components by a double membrane autophagosome fusing to the lysosome. Selective autophagy, like ER-phagy, permits the degradation of specific targets. This process generally involves specific cargo receptors containing LIR or GIM domains targeting bound cargo to the autophagosome through interactions with LC3 or GABARAP, respectively. FAM134B was the first ER-phagy receptor discovered. Loss of FAM134B can sensitize cells to apoptosis when challenged by nutrient deprivation or ER stress stimuli. CCPG1 is another ER-phagy cargo receptor that associates with FIP200, a component of the ULK1 complex facilitating ER trafficking to autophagosomes. Importantly, CCPG1 is transcriptionally regulated by ER stress. Taken together, signaling from the UPR and ER-phagy help regulate ER homeostasis. Defects in this process may contribute to pathological conditions, including metabolic and neurological disorders, cancer, and defense against infectious diseases (3).
  1. Sano, R. and Reed, J.C. (2013) Biochim Biophys Acta 1833, 3460-3470.
  2. Ferro-Novick, S. et al. (2021) Trends Biochem Sci 46, 630-639.
  3. Hübner, C.A. and Dikic, I. (2020) Cell Death Differ 27, 833-842.

Pathways & Proteins

Explore pathways + proteins related to this product.

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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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