RIPA buffer was used to extract total protein. Proteins were quantified using a BCA protein determination Kit (KeyGEN Biotech, Nanjing, China). 30 μg protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h. The incubation of blots was conducted with antibodies at 4 °C overnight: Bax (ab32503, 1:1000, Abcam, Cambridge, MA, USA), Bcl-2 (ab117115, 1:1000, Abcam), ILF3 (ab225626, 1:1000, Abcam), AURKA (ab108353, 1:1000, Abcam), E2F1 (ab4070, 1:500, Abcam), E-cadherin (ab231303, 1:1000, Abcam), Vimentin (ab92547, 1:1000, Abcam), PI3K (#3811, 1:1000, CST, Danvers, MA, USA), phosphorylated PI3K (p-PI3K, #4228, 1:1,000, CST), AKT (#9272, 1:1,000, CST), phosphorylated AKT (p-AKT, #9271, 1:1000, CST) or GAPDH (ab8245, ab9485, 1:5000, Abcam) and then with HRP-conjugated second antibody (#7074, 1:1000, CST). Protein bands were detected with ECL Plus reagent (Pharmacia, Piscataway, USA), and visualized using a Gel Imaging System. Bands were then quantified using ImageJ software (National Institutes of Health). The expression of GAPDH was used for data normalization.
Ab117115
Manufactured by Abcam
Sourced in United Kingdom
Ab117115 is a primary antibody that recognizes the human GAPDH protein. It is a mouse monoclonal antibody produced using a recombinant protein fragment as the immunogen.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab8245, by Abcam (2 mentions)
Ab97051, by Abcam (2 mentions)
Ab3191, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab225626, by Abcam (1 mentions)
Ab108353, by Abcam (1 mentions)
Ab4070, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Bca protein determination kit, by Keygen Biotech (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab203119, by Abcam (1 mentions)
Ab209857, by Abcam (1 mentions)
Ab28422, by Abcam (1 mentions)
Ab216494, by Abcam (1 mentions)
Ab90437, by Abcam (1 mentions)
14 protocols using ab117115
1
Western Blot Analysis of Apoptosis and EMT Markers
2
Protein Expression Analysis of Apoptosis and UPR Pathways
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The total protein was extracted by the modified RIPA buffer (P0013B; Beyotime, Shanghai, China), and the concentration was quantitated by the BCA protein assay kit (P0010; Beyotime, Shanghai, China). Then, the total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, and then the membranes were blocked in 5% skim milk diluted with TBST for 1 h at room temperature and probed with primary antibodies, including MFG-E8 (R&D Systems, AF2805, 1 : 500), BCL2 (Abcam, ab117115, 1 : 500), BAX (Abcam, ab216494, 1 : 500), cleaved caspase-3 (Abcam, ab90437, 1 : 500), GRP-78 (Abcam, ab230508, 1 : 500), XBP-1 (Abcam, ab230508, 1 : 500), ATF-6 (Abcam, ab203119, 1 : 500), ATF-4 (Abcam, ab23760, 1 : 500), CHOP (Abcam, ab23760, 1 : 500), p-PI3K (Abcam, ab125633, 1 : 500), PI3K (Abcam, ab154583, 1 : 500), p-AKT (Abcam, ab18785, 1 : 1000), AKT (Abcam, ab28422, 1 : 500), and β-actin (Abcam, ab209857, 1 : 1000) at 4°C overnight. Then, the membranes were incubated with a secondary rabbit anti-rabbit antibody (1 : 1000) the next day at room temperature for 1 h. The ImageJ software was employed for quantitation of the immunoreactive bands.
3
Western Blot Analysis of Smad3, Bax, and Bcl2
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Protein was extracted from CC cells (1 × 107) seeded in a six-well plate by RIPA buffer (Thermo Fisher Scientific, CA, USA) according to the manufacturer’s instructions. Then BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to determine the concentration of protein. Equal amounts of protein samples were separated by 12 percent SDS-PAGE and were transferred into PVDF membranes. The membranes were blocked with skim milk for 1 h and then probed with primary antibodies anti‐Smad3 (1:1000, ab28379; Abcam, Cambridge, MA), Bax (1:5000, ab182733; Abcam, Cambridge, MA) and Bcl2 (1:5000, ab117115; Abcam, Cambridge, MA) incubated at 4 °C overnight. After washed three times with 0.1% tween‐PBS (TBST), the membranes were incubated by secondary HRP-conjugated antibody at 37 °C for an hour. GAPDH was used as a loading control. After rewashed three times by 0.1% TBST, the results were analyzed using an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL) and visualized using an imaging system (Bio-Rad, CA, USA). All measurements were performed at least three times.
4
Protein Expression Analysis by Western Blot
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Total protein was extracted, separated on SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membrane was blocked with 5% non-fat milk in TBS and then incubated with antibodies against CMTM5 (ab96077, Abcam), AKT (4691, CST), pAKT (4051, CST), PI3K (ab86714, Abcam), p21 (2946, CST), CyclinD1 (2978, CST), CyclinE (4129, CST), Bcl2 (ab117115, Abcam), Bax (AB32503, Abcam), Bad (ab32445, Abcam), cleaved caspase 3 (9661, CST), MMP2 (13132, CST), MMP9 (13667, CST) overnight at 4 °C. HRP-conjugated IgG antibody were treated at 37 °C for 1 h. The bands were then visualized using BioImaging Systems (UVP Inc., Upland, CA, USA).
5
PCDH9 Protein Expression Analysis
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Proteins were extracted from cell lines using sodium dodecyl sulfate lysis buffer (2% sodium dodecyl sulfate, 10% glycerol, 0.1 mM dithiothreitol, and 0.2 M Tris–HCl, pH 6.8). Protein samples (50 ug) were resolved by SDS–PAGE and analyzed by immunoblots. PCDH9 antibody was from abcam (1:1000, ab117115).
6
Liver Tissue Protein Analysis
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The liver tissues (20 mg) were homogenized by a homogenizer in 180 µL Radio-Immunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with 2 µL phenylmethanesulfonyl fluoride (PMSF). The tissue lysates further lysed were incubated on ice for 1 h and then centrifuged at 13,200 rpm at 4 °C for 5 min. The protein content was measured using the Pierce BCA protein assay kit (Thermo VK312556). An equal amount of protein per sample was collected and boiled for Western blotting analysis. Western blotting was performed as previously described [21 (link)]. The antibodies used in this work included Nrf2 (12721S, CST, Danvers, MA, USA), HO-1 (43966S, CST), β-actin (A1978, Sigma, MO, USA), NF-κB (8242S, CST), p-NF-κB (Ser536) (13346, CST), p-MAPK (3510, CST), Bcl2 (ab117115, Abcam, Cambridge, U.K.), and Bax (ab32503, Abcam). Positive signals were visualized using a chemiluminescence (ECL) analysis kit (170-5061, Bio-Rad, Hercules, CA, USA) and recorded with the Chemi Doc system (Bio-Rad, USA). The positive bands were quantified by densitometry using ImageJ software (Bethesda, Rockville, MD, USA) and normalized to the density of β-actin.
7
Immunohistochemical Analysis of MYC and BCL2
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Slides of formalin-fixed, paraffin-embedded tissues were stained with an anti-MYC antibody (Abcam, Y69, ab32072) and anti-BCL2 antibody (Abcam, Bcl2/100, ab117115) using a standard streptavidin peroxidase technique and chromogen diaminobenzidine. The staining pattern for MYC protein was distinctly nuclear, whereas staining for BCL2 protein showed a well-defined cytoplasmic staining pattern. The percentage of positive expression lymphoma cells was recorded and used for analysis. To make the determination of expression status more objective, the scoring was performed independently by two different pathologists. The interobserver reproducibility was assessed using the difference between the scorings of each observer. The mean of the scorings from each reader was then calculated and used for the statistical analysis.
8
Hypoxia-reoxygenation effects on hBMSCs
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hBMSC cultured in DMEM supplemented with 10% FBS and 10 ng/ml Rg1-loaded microspheres or blank microspheres were treated with hypoxia-reoxygenation. The cells were first incubated under hypoxia using a 95% N2 and 5% CO2 for 5 h at 37°C in a water-jacketed N2/CO2 incubator and then incubated in a standard incubator with 5% CO2 in normal air at 37°C for 20 h. Then, the cells were harvested and washed with ice-cold PBS three times, lysed with ice-cold RIPA lysis buffer (Beyotime, China) with 1 mmol/l PMSF. The protein concentrations were tested by BCA Protein Assay Kit (Thermo Fisher Scientific, U.S.A.). Twenty five micrograms of extracted protein were separated by SDS/PAGE (12% (w/v) gel) and transferred to PVDF membranes (Thermo Fisher Scientific, U.S.A.). The membrane was blocked with 5% nonfat dry milk in 0.1% Tween-20 (TBST) at room temperature for 1 h and immunoblotted with anti-Bax (Abcam, ab32503) and anti-Bcl-2 (Abcam, ab117115) at 4°C overnight, followed by incubation with the appropriate horseradish peroxidase–conjugated secondary antibodies for another 1 h at room temperature. After washing five times with PBS, the blots were treated with enhanced chemiluminescent substrate (Thermo Fisher Scientific, U.S.A.) and then exposed to the ChemiDoc MP system (Bio–Rad Laboratories, U.S.A.). Densitometry was performed using Image Lab Software and normalized to GAPDH control.
9
Western Blot Analysis of Liver Proteins
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Proteins obtained from liver tissues were analyzed by Western blot analysis. Blots underwent incubation with antibodies against JNK (Anti-JNK1/2/3 antibody bs-2592R; Bioss, Beijing, China), phosphorylated-JNK (P-JNK) (Anti-phospho-JNK1/2/3 antibody bs-1640R; Bioss), B-cell lymphoma-2 (Bcl-2) (Anti-Bcl-2 antibody ab117115; Abcam Inc., Cambridge, MA, UK), Bcl-2 associated protein X (Bax) (Anti-Bax antibody bs-0127R; Bioss), Cleaved Caspase-3 (Anti-Cleaved Caspase-3 ab49822; Abcam), Cytochrome C (Anti-Cytochrome C antibody ab90529; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Anti-GAPDH antibody ab8245; Abcam). The blots were scanned with ChemiDoc™ XRS (Bio-Rad, USA). Image J 7.0 software was used for the analysis of the gray area density.
10
Western Blot Analysis of Apoptosis Markers
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Cells were washed with PBS and lysed on ice in RIPA buffer (V900854, Sigma) containing a cocktail of protease inhibitors (P8340, Sigma) following the manufacturers protocols. Protein concentration was measured using the BCA assay. The protein fractions were suspended in loading buffer and denatured at 100°C for 5 min. Total proteins (20 µg/lane) were separated on 12% SDS-PAGE and transferred to PVDF membranes, which were blocked in 5% fat free milk in TBST buffer (0.1% Tween-20) for 2 h at room temperature. BRCA1 levels were analyzed using a mouse monoclonal anti-BRCA1 antibody (1:1,000; ab16780; Abcam); Bcl-2, Bax, pro-caspase-3, cleaved-caspase-3, and cytochrome c levels were detected using the following mouse or rabbit monoclonal antibodies at a dilution of 1:1,000: ab117115, ab5714, ab13586, ab32042, and ab13575, respectively (Abcam). The secondary antibodies used were goat anti-rabbit antibody (1:1,000; Sc2030; Santa Cruz Biotechnology) and rabbit anti-mouse antibody (1:1,000; Sc358917; Santa Cruz Biotechnology). GAPDH was used as the endogenous control to normalize the expression levels of the proteins of interest. GAPDH levels were detected using HRP-conjugated mouse anti-GAPDH monoclonal antibody (1:5,000; HRP-60004, Proteintech). The densitometry scan of the western blotting was performed by Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
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