Primary antibodies for IPF lung tissue include ATF6 (Abcam, ab37149), PERK (Abcam, ab229912), IRE1 (Abcam, ab37073), Grp78 (Abcam, ab21685), CHOP (Abcam, ab11419), α-SMA (Abcam, ab119952), and Collagen I antibody (Abcam, ab138492). For mouse LR-MSC, they include ATF6 (Abcam, ab37149), PERK (Abcam, ab229912), IRE1 (Abcam, ab37073), Grp78 (Abcam, ab21685), CHOP (Abcam, ab11419), α-SMA (Abcam, ab119952), Collagen I antibody (Abcam, ab21286), anti-Smad2/3 (Abcam, ab202445), anti-pSmad2 (Abcam, ab188334), anti-pSmad3 (Abcam, ab529035), anti-Wnt10a (Abcam, ab106522), and anti-beta Catenin (Abcam, ab32572). Anti-Lamin B1 (Abcam, ab16048) was used for loading control of nuclear protein. Anti-GAPDH (Abcam, ab8245) was used for loading control for total protein.
Ab37073
Manufactured by Abcam
Sourced in United States, China
Ab37073 is a primary antibody that recognizes the protein of interest. It is suitable for use in various immunoassays, including Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Ab48187, by Abcam (10 mentions)
Ab21685, by Abcam (4 mentions)
Ab37149, by Abcam (4 mentions)
Ab13847, by Abcam (4 mentions)
Ab11419, by Abcam (3 mentions)
Ab203119, by Abcam (3 mentions)
Ab66579, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Ab179461, by Abcam (2 mentions)
Ab79483, by Abcam (2 mentions)
Ab229912, by Abcam (1 mentions)
Ab202445, by Abcam (1 mentions)
Ab106522, by Abcam (1 mentions)
Ab21286, by Abcam (1 mentions)
Ab119952, by Abcam (1 mentions)
Ab188334, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
23 protocols using ab37073
1
Comprehensive Antibody Panel for IPF and LR-MSC
2
Revealing ER Stress and Autophagy Markers
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Following the desired treatments, CHO cells were washed and harvested on ice, and then lyzed with ice-cold protein extraction buffer (Beyotime, Nanjing, China). The protein concentration was determined using BCA protein assay kit (ComWin, Beijing, China). Equal amounts of protein were loaded into gel wells, separated by electrophoresis on SDS-polyacrylamide gels and transferred onto PVDF transfer membrane (Millipore, Bedford, MA). The transferred blots were blocked with blocking agents (5% w/v BSA and 0.05% Tween-20 in TBS) for 1 h at room temperature. Blots were then incubated in a certain proportion of specific primary antibodies: anti-GRP78 (ab25192 Rabbit monoclonal, Abcam), anti-ATF-6 (ab203119 Rabbit polyclonal, Abcam), anti-PERK (C33E10 Rabbit polyclonal, Abcam), anti-IRE1 (ab37073 Rabbit polyclonal, Abcam), anti-CHOP (ab11419 Mouse monoclonal, Abcam), anti-Beclin1 (ab55878 Rabbit polyclonal, Abcam), anti-LC3B (ab81785 Rabbit polyclonal, Abcam) (1:1000) overnight at 4°C. These membranes were rinsed 3 times for 10 min each with TBS-Tween (Sigma, St. Louis, MO, USA) and incubated with HRP conjugated secondary antibodies (1:5000) (CWBio, Beijing, China). After washing, protein bands were visualized by an enhanced chemiluminescence detection kit (ComWin, Beijing, China). Each band density was quantified using Image J software (GE Healthcare, Piscataway, New Jersey, USA).
3
Quantifying Endoplasmic Reticulum Stress Response
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The aorta tissue or RAW264.7 macrophages were lysed for 30 min at 4°C in lysis buffer. Total protein concentrations were determined using bicinchoninic acid reagent. Total protein (50–100 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were incubated with anti-IRE1α (ab37073; 1:800; Abcam), anti-glucose-regulated protein 78 (GRP78; 11587-1-AP; 1:500; ProteinTech, Wuhan, China), anti-p-IRE1α (Ser724; ab48187; 1:800; Abcam), IκB kinase α (IKKα; 2682S; Cell Signaling Technology, Danvers, MA, USA), IκB kinase β (IKKβ; 2684S; Cell Signaling Technology), anti-p-IκB kinase α/β (p-IKK; Ser176/180; 2697T; 1:800; Cell Signaling Technology), IκB (YM3718; 1:500; ImmunoWay, Newark, DE, USA), anti-NF-κB p65 (YT5339; 1:500; ImmunoWay), anti-β-actin (6008-1-Ig; 1:750; ProteinTech) and anti-LaminB (sc-6216; 1:500; ZSGB-Bio) primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies (ZB-2301; ZSGB-Bio) were applied the following day for 2 h at 37°C. The bands were visualized using chemiluminescence (Beyotime, Shanghai, China). Band density was analyzed using fusion software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to β-actin or Lamin B density.
4
ER Stress Pathway Molecular Markers
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The primary antibodies used are listed as follows: anti-cytochrome c (1:1,000, 10993-1-AP, Proteintech, rosemont, IL, USA), anti-caspase-9 (1:1,000, #9508, Cell Signaling, Danvers, MA, USA), anti-PERK (1:1,000, #3192, Cell Signaling, Danvers, MA, USA), anti-phosphorylated-PERK (anti-p-PERK; Thr982;1:800, DF7576, Affinity Biosciences, Cincinnati, OH, USA), anti-IRE1 (1:500, ab37073, Abcam, Cambridge, MA, USA), anti-p-IRE1 (phosphor S724; 1:1,000, ab48187, Abcam, Cambridge, MA, USA), anti-activating transcription factor 6 (anti-ATF6; 1:1,000, ab203119, Abcam, Cambridge, MA, USA), anti-N’ATF6 (1:800, ab37149, Abcam, Cambridge, MA, USA) and anti-β-actin (1:3,000, GTX109639, GeneTex, Irvine, CA, USA). The ER stress activator Tunicamycin (TM) and the p-PERK inhibitor GSK2656157 were obtained from MedChem Express (Monmouth Junction, NJ, USA). The p-IRE1 inhibitor 3,5-dibromosalicylaldehyde (DBSA) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan).
5
Immunofluorescence Analysis of Myocardial Cells
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The treated myocardiocytes were cultured in 6-well plates for 12 h at 37°C, washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and then incubated with 0.1% Triton X-100 and 1% BSA for 30 min at room temperature. Myocardiocytes were incubated with primary anti-mouse antibodies against Bim (1:1,000), CHOP (1:1,000), activating transcription factor 4 (ATF4; 1:1,000; product code ab216839; Abcam), inositol-requiring enzyme 1α (IRE1α; 1:1,000; product code ab37073; Abcam), thiobarbituric acid reactive substances (TBARS; 1:1,000; ab118970; Abcam), reactive oxygen species (ROS; 1:1,000; ab186027; Abcam), H2O2 (1:1,000; ab138874; Abcam) overnight at 4°C. Myocardiocytes were washed with PBS and incubated with corresponding anti-rabbit secondary antibody (1:2,000, product code ab7090; Abcam) for 2 h at 25°C. Subsequently, myocardiocytes were stained with 5% DAPI for 30 min at room temperature. Images of myocardiocytes were captured using a Zeiss confocal spectral microscope (Carl Zeiss AG) at a magnification of ×100.
6
Hypothalamic ER Stress Signaling Proteins
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A subset of 6 mice per group were used for assessment of hypothalamic PERK, pPERKThr980, eIF2α, peIF2αSer55, IRE1α, pIRE1αSer724, ATF6, TNFα and tubulin protein expression using standard methodology, as previously described (McGavigan et al., 2015 (link)). Antibodies against PERK, pPERKThr980 and peIF2αSer55 were from Cell Signaling (3179 and 3398, respectively). Antibodies against eIF2α, ATF6 and tubulin were from Santa Cruz (sc-133227, sc-22799, sc-23948, respectively) and TNFα, IRE1α and pIRE1αSer724 antibodies were from Abcam (ab66579, ab37073, ab104157, respectively). All antibodies were used at a concentration of 1:1000 and pixel densities of immunoreactive bands were quantified using FluorChem 9900 (Alpha Innotech, CA).
7
Immunoblotting analysis of signaling proteins
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Immunoblotting was performed as previously described [16 (link), 18 (link)]. Primary antibodies were obtained from Santa Cruz Biotechnology: α tubulin, 1:10000, sc-23948; BiP, 1:1000, sc-13968; XBP1, 1:1000, sc-7160; Akt, 1:5000, sc-8312; Cell Signaling Technology: pAkt, 1:5000, S473 or Abcam: IRE, 1:2000, ab37073; pIRE, 1:1000, ab48187; TNFα 1:5000 ab66579. Chemiluminescence was performed with Luminata Forte Western HRP Substrate (Millipore) and detected using Chemidoc MP Imaging system. Protein expression was quantified using ImageLab 5.1 software.
8
Immunofluorescence Staining of Brain Tissue
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Immunofluorescence staining was performed as previously described (Shi et al., 2017 (link)). Tissue was mounted in slides and washed with 0.1 M PBS three times for 5 min, following which it was incubated in 0.3% Triton X-100 in 0.1 M PBS for 30 min at room temperature. Slices were then washed using 0.1 M PBS for 5 min, three times, and the primary antibodies were applied overnight at 4°C: anti-BI-1 (1:100, ab18852, Abcam), anti-IRE1α or anti-pIRE1α (1:100, ab48187 and ab37073, Abcam), anti-NeuN (1:100, ab104224, Abcam), anti-GFAP (1:100, ab53554, Abcam), anti-Iba1 (1:100, ab5076, Abcam), anti-CD11b/c (1:100, ab1211, Abcam) and anti-CC3 (1:100, 9661S, Abcam). After washing with PBS, sections were incubated with the appropriate secondary antibodies: anti-rabbit IgG-TR (711-295-152), anti-mouse IgG-FITC (715-095-150), anti-goat IgG-FITC (705-095-008), anti-rabbit IgG-FITC (711-095-152) (all at 1:200, Jackson ImmunoResearch) for 1 h at room temperature. Finally, slides were covered with DAPI and visualized using a fluorescence microscope. To analyze the images, Magna Fire SP system (Olympus) was used.
9
Western Blot Analysis of Irisin and Related Proteins
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Western blot analysis was performed as described previously.22 PVDF membranes were incubated with primary rabbit anti‐irisin antibody (1:1000 dilution, ab174833; Abcam); anti‐claudin‐1 antibody (1:1000 dilution, 13050‐1‐AP; Proteintech); rabbit anti‐occludin antibody (1:1000 dilution, ab216327; Abcam); rabbit anti‐AMPKα antibody (1:1000 dilution; Cell Signaling Technology); rabbit anti‐PAMPKα antibody (1:1000 dilution; Cell Signaling Technology); rabbit anti‐UCP 2 antibody (1:1000 dilution, ab203244; Abcam); rabbit anti‐UCP1 antibody (1:1000 dilution, 14670S; Cell Signaling Technology); rabbit anti‐IRE1 antibody (1:1000 dilution, ab37073; Abcam); rabbit anti‐CHOP antibody (1:1000 dilution, 5554; Cell Signaling Technology); or mouse anti‐β‐actin monoclonal antibody (1:1000 dilution, HRP‐60008; Proteintech) overnight at 4°C. Then, secondary HRP‐conjugated goat anti‐rabbit IgG (1:2000 dilution, SA00001‐2; Proteintech) was incubated for 1 hour at room temperature. Protein expression was detected by a chemiluminescence system (Bio‐Rad) and quantified by ImageJ2x software.
10
Western Blot Analysis of UPR Markers
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Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) and the concentration of protein samples was determined via BCA (Beyotime, Shanghai, China) methods. Thereafter, protein samples were separated by 10% SDS-PAGE gel (Beyotime, Shanghai, China) and transferred to PVDF membranes (Millipore, MA, USA). PVDF membranes were blocked in 5% skim milk powder for 1 h and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this research were IRE-1 (ABCAm, ab37073, 1:2000), CHOP (CST, #5554, 1:1000), XBP-1 U (ABCAm, ab37152, 1:2000), XBP-1S (CST, #40,435, 1:1000), Caspase-12 (ABCAm, ab62484, 1:1000), Bax (ABCAm, ab32503,1:5000), Bcl-2 (ABCAm, ab32124, 1:1000), Caspase-3 (ABCAm, ab13847, 1:500), Cleaved caspase-3 (ABCAm, ab2302, 1:500) and GAPDH (ABCAm, ab9484, 1:1000). On the second day, PVDF membranes were washed with PBST for three times and incubated with second antibodies for 2 h at room temperature. At last, protein signals were developed with enhanced chemiluminescence reagents (Pierce, IL, USA) and analyzed with Image J software.
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