Rat anti-mouse F4/80 antibody was purchased from Serotec; Rat anti-mouse CD31 was purchased from Dianova (Hamburg, Germany). Anti-actin and anti-smooth muscle actin (SMA) monoclonal antibodies were purchased from Sigma (St. Louis, MO). Antibodies directed against phopsho-AMPK alpha, phospho-p38 and phospho-JNK, histone H3 and H3K4-di (H3K4me2) and -tri (H3K4Me3) methylation, H3K27 tri-methylation and acetylated H2BK5 were purchased from Cell Signaling Technology (Beverly, MA). Anti-WDR5, anti-BiP, and anti-Ly6g antibodies were purchased from Abcam (Cambridge, UK). The selective inhibitors of LSD1 (GSK-2879552), WDR5 (OICR-9429), MKL1 (CCG-203971), CXCR2 (SB 225002), p38 (SB208530), and JNK (sp600125) were purchased from ApexBio (Boston, MA) and were dissolved in DMSO as stock solutions. DMSO (Sigma D2438) was added to the cell culture medium as control. The heparanase inhibitors H1001 and PG545 were kindly provided by HepaRx Ltd (Ness-Ziona, Israel) and Zucero Therapeutics (Darra, Queensland, Australia), respectively. Mouse CXCL2/MIP-2 Quantikine ELISA kit was purchased from R&D Systems (Minneapolis, MN). The MyD88 peptide inhibitory set was purchased from Novus Biologicals (Centennial, CO). Latent heparanase was purified from medium conditioned by CHO cells overexpressing heparanase essentially as described (25 ) and was added to cell cultures at 1 μg/ml.
Anti bip
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-BiP is an antibody that binds to the BiP (Binding Immunoglobulin Protein) protein. BiP is a molecular chaperone involved in the proper folding and assembly of proteins within the endoplasmic reticulum.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti v5, by Thermo Fisher Scientific (3 mentions)
Anti chop, by Abcam (2 mentions)
Anti giantin, by Abcam (2 mentions)
4 well chamber slide, by Thermo Fisher Scientific (2 mentions)
Anti flag m2, by Abcam (2 mentions)
Lsm image examiner, by Zeiss (2 mentions)
Anti pdi, by Cell Signaling Technology (2 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (2 mentions)
Anti tgn, by Abcam (2 mentions)
Anti chop, by Cell Signaling Technology (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Er tracker red, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Mouse cxcl2 mip 2 quantikine elisa kit, by R&D Systems (1 mentions)
Rat anti mouse f4 80 antibody, by Bio-Rad (1 mentions)
H3k4me2, by Cell Signaling Technology (1 mentions)
Anti wdr5, by Abcam (1 mentions)
Anti actin, by Merck Group (1 mentions)
16 protocols using anti bip
1
Antibody and Inhibitor Reagents for Assays
2
Antibody-Based Histology and Cell Staining Protocols
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The following antibodies for histology and cell staining were purchased from Santa Cruz Biotechnology: anti-MUC5AC (Santa Cruz, CA, sc-16903, AB_649616), anti-phospho-PI3K p85α (Tyr467: Santa Cruz, CA, sc-293115, AB_10844180), anti-PI3K p85α (Santa Cruz, CA, sc-31970, AB_2268186), anti-phospho-Akt1 (Thr308: Santa Cruz, CA, sc-135650, AB_2224730), anti-Akt1 (Santa Cruz, CA, sc-1618, AB_630849), anti-phospho-NFκB p65 (Ser536: Santa Cruz, CA, sc-33020, AB_2179018), anti-NFκB p65 (Santa Cruz, CA, sc-109, AB_632039), anti-Lyn (Santa Cruz, CA, sc-15, AB_2281450), and anti-β-actin (Santa Cruz, CA, sc-130656, AB_2223228). The following antibodies for histology were purchased from Abcam Biotechnology: anti-BIP (Abcam, ab21685, AB_2119834), anti-CHOP (Abcam, ab11419, AB_298023), anti-histone H3 (Abcam, ab1791, AB_302613), and anti-IL-13 (Abcam, ab133353, AB_11157609). Anti-phospho-Lyn (Tyr416: Cell Signaling Technology, #2101, AB_331697) was purchased from Cell Signaling Technology. Anti-IL13 (R&D Systems, AF-413-NA, and AB_2124173) and IL-13 ELISA reagents (R&D Systems, M1300CB) were purchased from R&D Systems. IL-13 (PeproTech, #200-13), 4-Phenylbutyric acid (4-PBA, Sigma-Aldrich, P21005), PI3K Inhibitor PI-103 (Selleck, S1038) were purchased as indicated. A nonsilencing siRNA control and a Lyn-specific siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
3
AHA-Labeling and Biotin Enrichment of Newly Synthesized Proteins
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To assess the effect of 4E-BP overexpression, 4E-BPLLAA was induced in S2 cells stably transfected with pMT-4E-BPLLAA with 0.5mM copper sulfate for 2 h in methionine-free Schneider’s medium to enable AHA uptake. The cells were then labeled with 4 mM AHA (Anaspec) for 1 h. AHA-labeled proteins in the extracts were clicked to biotin-PEG4 alkyne (Invitrogen) using the manufacturer’s protocols. AHA-conjugated peptides were further enriched by incubation with Streptavidin-agarose (Thermo Fisher Scientific) followed by rigorous washing with PBS-Tween (0.5%). The bound fraction was collected by boiling the beads in sample buffer and analyzed by Western blotting with Streptavidin-IRDye 800 (Rockland Immunochemicals), guinea pig anti-BiP (1:1,000), mouse anti-Hsp70 (Abcam), mouse antiactin (EMD Millipore), and mouse antitubulin (Abcam).
4
Molecular Mechanisms of Neuroinflammation
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Antibodies involved in this study contain: anti-Cleaved caspase3 (Abcam, ab32042); anti-HMGB1 (Abcam, ab79823); anti-TNF-α (Abcam, ab183218); anti-IL-1β (Abcam, ab9722); anti-CCL2 (Abcam, ab25124); anti-Bip (Abcam, ab21685); anti-CHOP (Abcam, ab11419); anti-HO-1 (Abcam, ab52947); anti-MANF (Abcam, ab67271); anti-GAPDH (Abcam, ab3285); anti-HIF-1α (Abcam, ab243860); Anti-CD163 (Abcam, ab182422); Goat Anti-Rabbit IgG H&L (HRP) (Abcam, ab6721); PE anti-CD11b (Abcam, ab25533); APC anti-Ly6C (Abcam, ab93550); Alexa Flour 488 anti-Ly6G (Abcam, ab283276). The involved reagents contain: DNFB (Sigma, St Louis, MO, USA, 42085); hrMANF protein (Abcam, ab123227); Goat Anti-Mouse/Rabbit Polymer Immunohistochemistry Detection Kit (ZSGB-BIO, PV-6000); Lipofectamine™ 3000 (Thermo Fisher, L3000150).
5
Immunofluorescence Staining of Cellular Proteins
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HCT-116 or SW620 stable cells were grown in 4-well chamber slides (Thermo Fisher Scientific) and allowed to attach for 1 day. Cells were fixed and permeabilized with 100% methanol (prechilled at − 20 °C) at room temperature for 5 min and blocked with 1% bovine serum albumin for 1 h. Then, the cells were incubated with anti-FLAG M2, anti-BiP (Abcam), or anti-giantin (Abcam) antibodies. After washing cells with phosphate-buffered saline (PBS), they were incubated with secondary Alexa Fluor® 488-conjugated rabbit anti-mouse IgG or Alexa Fluor® 594-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) antibodies. Nucleic acids were stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories). Images were acquired using a confocal laser-scanning microscope and analyzed using an LSM image examiner (Carl Zeiss).
6
Comprehensive Antibody Validation Protocol
7
Immunofluorescence Subcellular Localization
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Cells were cultured on Lab-Tek chamber slides (Thermo Fisher Scientific) and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS containing 1% bovine serum albumin (BSA) for 5 min, and incubated with each antibody: anti-V5 (Thermo Fisher Scientific), anti-FLAG (Sigma), anti-BIP (Abcam, Cambridge, MA, USA), and anti-TGN (Abcam). ER-Tracker™ Red (Thermo Fisher Scientific) and the Golgi tracker CellLight® Golgi-RFP (Thermo Fisher Scientific) were used according to the manufacturer’s instructions. Fluorescence was monitored with a TCS SP2 AOBS inverted spectral confocal scanning system (Leica, Wetzlar, Germany) with an oil immersion 63× objective after secondary staining with Alexa Fluor 488-conjugated F(ab’)2 fragment of goat anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor 594-conjugated F(ab’)2 fragment of goat anti-rabbit IgG (Thermo Fisher Scientific).
8
Western Blot Analysis of Protein Expression
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Protein extracts were prepared by lysing cells in RIPA buffer. Protein yield was quantified using the Bio-Rad DC protein assay kit (Bio-Rad, USA). Equal amounts of protein were separated by 10% SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) before wet-transfer onto PVDF membrane (Amersham Biosciences, UK). Nonspecific binding sites were blocked by overnight incubation with 5% nonfat dry milk in Tris-buffered saline with 1% Tween (TBS-T; 130 mmol/L NaCl, 20 mmol/L Tris, pH7.6 and 1% Tween). Primary antibodies used were anti-HSC70 (Abcam, UK), anti-BiP, anti-Calnexin, anti-ERo1α, anti-CHOP, anti-PERK, anti-PDI, anti-LC3B, anti-COX2 (Cell Signaling, USA) and β-ACTIN (Abcam, UK) which was used as a loading control. All primary antibodies were diluted 1:1000; except for the anti-β-ACTIN, which was diluted 1:100,000. Full length scans are presented as supplementary information . Note that some images were reflected for consistency in the sequence of presentation.
9
Quantitative Western Blot Analysis of Protein Levels
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Disease-relevant tissues, including DRG, stomach, and colon, were homogenized on ice using a small glass rod homogenizer in lysis buffer (as in the section ‘Evaluation of Cell Toxicity Induced by TTR Assemblies and its Inhibition’). After centrifugation at 18,700 g for 20 mins at 4 °C, protein concentration in the supernatant was determined by the Bradford protein assay (Bio-Rad). Fifty micrograms of total protein from each tissue sample was fractionated on 15 % sodium dodecyl sulfate-PAGE and transferred onto nitrocellulose Hybond-C membranes using the Mini Trans-Blot Cell (Bio-Rad) system. The primary antibodies and the respective dilutions used were as follows: rabbit polyclonal anti-TTR (1:1000; Dako, Carpinteria, CA, USA); rabbit polyclonal anti-BiP (1:1000) (Abcam); mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000) (Abcam). Blots were visualized using enhanced chemiluminescence (Amersham ECL Prime) and quantified as described in ‘Detection of TTR Aggregation in Cell Culture Medium by a Dot-blot Filter Assay’. Immunosignals were normalized to GAPDH expression. Each group was compared with control. Results are presented as normalized density ± SD. A p-value < 0.05 was considered statistically significant.
10
Western Blot Analysis of Cellular Proteins
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Cellular proteins were extracted using cold RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail. Total proteins (30–50 μg) were separated on 10 or 12% SDS-polyacrylamide gels and then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dried milk, and then incubated with primary antibodies (anti-FLAG, 1:1000, Cell Signaling Technology; anti-BiP, 1: 1000, Abcam; anti-cleaved Caspase-3, 1:1000, Cell Signaling Technology; anti-beta-Actin-Peroxidase, 1:5000, Sigma-Aldrich) overnight at 4°C, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1000, Cell Signaling Technology). Chemiluminescence solution (MilliporeSigma, United States) was used to reveal the bands. Representative blots from at least three independent experiments were shown.
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