RIPA buffer (Solarbio, Shanghai, China) was used to lyse cells and a BCA kit (Beyotime, Shanghai, China) was used to quantify protein levels. The protein concentration was about 50 mg/mL, which was separated by 10% SDS-PAGE gel. β-actin (1:5000, AbMART, Shanghai, China, M2009) was used as a loading control. The antibody Fam98b (1:2000, FineTest, Wuhan, China, FNab03001), α-SMA (1:2000, Abcam, Cambridge, UK, ab124964), Collagen I (1:2000, Abcam, Cambridge, UK, sc-59772), Smad2/3 (1:2000, Abcam, Cambridge, UK, ab63672), BAD (1:2000, AbMART, Shanghai, China, 67830-1-Ig), BAX (1:2000, AbMART, Shanghai, China, T40051), Bcl2 (1:2000, AbMART, Shanghai, China, T40056), TGFβ (1:500, Wanleibio, Shenyang, China, WL02998), P-Smad2/3 (1:1000, Absin, Shanghai, China, abs131873), NOTCH3 (1:1000, Proteintech, Wuahn, China, 55114-1-AP) were used as primary antibody. The secondary antibodies were obtained from Invitrogen (1:20,000, Thermofisher, SA5-35521, SA5-35571). An Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) was employed to scan the blots. The quantitative analysis of grey values was performed using Image J software (NIH, USA).
Bcl2 associated x (bax)
Manufactured by Abmart
Sourced in China
Bax is a versatile laboratory equipment designed to perform a range of scientific procedures. It is a compact and efficient instrument that can be used for various applications in research and diagnostic settings. The core function of Bax is to provide a controlled and consistent environment for the analysis and processing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Abmart (7 mentions)
Caspase 3, by Abmart (3 mentions)
Gapdh, by Proteintech (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Caspase 9, by Abmart (2 mentions)
Odyssey two color infrared laser imaging system, by LI COR (1 mentions)
Sa5 35521, by Thermo Fisher Scientific (1 mentions)
Notch3, by Proteintech (1 mentions)
α sma, by Abcam (1 mentions)
Smad2 3, by Abcam (1 mentions)
Collagen 1, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
β actin, by Abmart (1 mentions)
β actin, by OriGene (1 mentions)
Ab52866, by Abcam (1 mentions)
Electrochemiluminescence, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gapdh af0006, by Beyotime (1 mentions)
Anti mouse immunoglobulin g igg, by Santa Cruz Biotechnology (1 mentions)
9 protocols using bcl2 associated x (bax)
1
Protein Expression Analysis in Cells
2
Apoptosis Pathway Regulation Assay
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Reagents used in this study were Fetal bovine serum (S-FBS-SA-015, SERANA company), DMEM high glucose medium (batch number 12100, SOLAIBAO), thiazole blue powder (batch number 298-93-1, SOLAIBAO), RNA extraction kit (batch number ER501-01, Beijing all-gold biological). A two-step fluorescence quantitative PCR kit (batch number AUQ-01, Beijing all-gold biology) RIPA tissue/cell lysate (batch number R0010, Suolaibao), BCA protein detection kit (batch number PC0020, Suolaibao), CASP3 (batch number ET1608-64, HUABIO), BAX (batch number T40051F, Abmart), β-actin (batch number TA811000, ORIGENE), HRP-labeled rabbit anti-mouse antibody (batch number WLA024a, ten thousand kinds of biology), HRP-labeled goat anti-rabbit antibody (batch number HA1031, HUABIO), 4× protein loading buffer (including DTT) (batch number P1015, Suolaibao), skim milk powder (batch number D8340, Suolaibao), ECL ultrasensitive chemiluminescence solution I (batch number KF005. ECL Hypersensitive Chemiluminescence Solution II (lot number KF001, Solebold).
3
Western Blot Analysis of Protein Expression
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Total protein was extracted from cell lines using lysis buffer (Thermo Fisher Scientific), containing a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China), and quantified using the Bradford method. Next, 40 µg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (8%), and the separated proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with primary antibodies against: TRAF4 (H2818, 1:100, Santa Cruz Biotechnology), Eg5 (1:1000), Smurf2 (F0641, 1:100, Santa Cruz Biotechnology), HA (TA180128S, 1:1000, Origene), α-tubulin (ab52866, 1:1000, Abcam), Caspase-3 (M005851F, 1:1000, Abmart), Bcl-2 (T40056F, 1:1000, Abmart), Bax (T40051F, 1:1000, Abmart), Ki67 (550609, 1:1000, BD Pharmingen), GAPDH (AF0006, 1:2000, Beyotime) and β-actin (1:2000, Beyotime). Next, the membranes were incubated with secondary HRP-conjugated antibody, anti-mouse immunoglobulin G (IgG), or anti-rabbit immunoglobulin (1:2000, Santa Cruz Biotechnology) at 37°C for 2h. Finally, antibody binding wasvisualizedusing electro-chemiluminescence (Thermo Fisher Scientific), and quantified using ImageJ (National Institute of Health, Bethesda, MD, USA).
4
Protein Expression Analysis in Cellular Processes
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Western blot and RT-qPCR were well established previously18 (link). The primary antibodies used in this study are listed as follows: PDK4 (1:1000, 1:100; Abclone), BCL2 (1:1000; Abmart), BAX (1:1000; Abmart), Caspase3 (1:1000; Proteintech Group), E-cad (1:1000; Proteintech Group), ZO-1 (1:1000; Abmart), α-SMA (1:1000; Proteintech Group), GAPDH (1:1000; Proteintech Group), and β-tubulin (1:1000; Abmart), GAPDH and β-tubulin were used as the loading control. The original, unprocessed versions of western blots images are shown in the Supplementary Figure 1 . The primers sequences used are listed in Supplementary Table 1 .
5
Western Blot Analysis of Apoptotic Pathways
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Proteins were extracted from HaCaT cells, then after 8 or 10% SDS-PAGE resolved, transferred to PVDF membranes (EMD Millipore). After blocking membranes using 5% nonfat dry milk, was incubated overnight with primary antibodies against Bax, Caspase 8, Caspase 9 (1:1000; Abmart); Caspase 3, Bcl-2, p-mTOR/mTOR, p-P38/P38, β-actin, p-P44/42/P44/42, and GAPDH (1:1000; CST); p-PI3K/PI3K (1:500; Abcam). We used HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:8000; Abmart Biological Reagents Co., Ltd.) as the secondary antibodies to label the antibodies above.
6
Western Blotting Analysis of Apoptosis Mediators
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In this study, the following antibodies were employed: BCL-2 (T40056T40056; Abmart, Shanghai, China), BAX (T40051; Abmart), Cytochrome C (T55734T55734; Abmart), PARP (T40050; Abmart), Caspase 3 (T40044; Abmart), Caspase 9 (T40046; Abmart), RIG-I (#20566-1-AP; Proteintech, Wuhan, China), mitochondrial antiviral signaling protein (MAVS, #66911-1-Ig; Proteintech), IRF3 (#11312-1-AP; Proteintech), P-IRF3 (#29528-1-AP; Proteintech), and GAPDH (#60004-1-Ig; Proteintech). Cells were harvested and lysed in radio-immunoprecipitation assay buffer (BL504A; Biosharp, Tallinn, Estonia) supplemented with Halt protease (BS-00-0903; Biosharp) and phosphatase inhibitor cocktail (BL615A; Biosharp) on ice. Subsequently, the cell lysates underwent Western blotting analysis, with GAPDH serving as a loading control.
7
Western Blot Analysis of Autophagy and Apoptosis Markers
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Cells and tissues were lysed in RIPA buffer supplemented with 1% PMSF (Solarbio, Beijing, China). Protein concentrations were quantified with a BCA Protein Assay Kit (Beyotime, Shanghai, China). The proteins were separated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Boston, MA, USA). Then, the membranes were incubated with the following primary antibodies overnight at 4 °C: ATP6V1B1 (1:1000, Proteintech, Wuhan, China), β-actin (1:1000, Proteintech, Wuhan, China), GAPDH (1:10,000, Proteintech, Wuhan, China), mTOR/p-mTOR (1:1000, Abmart, Shanghai, China), P62 (1:2000, Abmart, Shanghai, China), LC3B (1:2000, Abcam, Shanghai, China), Bax (1:1000, Abmart, Shanghai, China), Bcl2 (1:1000, Abmart, Shanghai, China), Caspase-3 (1:1000, Abmart, Shanghai, China), and Cleaved Caspase-3 (1:1000, Abmart, Shanghai, China). The following day, the membranes were incubated with an IgG secondary antibody (1:10,000, Invitrogen, USA) at room temperature. Signal visualization was achieved using an Odyssey CLx (LI-COR, USA).
8
Protein Inhibitor-Based Cell Signaling
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Sigma (Sigma-Aldrich, Shanghai, China) provided the p53 inhibitor Pifithrin-α (PFT-α), while Merck (Merck KGaA, Darmstad, Germany) provided the p38 MAPK inhibitor SB203580 and the SAPK/JNK inhibitor SP600125. Anti-p53, p38 MAPK, JNK/SAPK, Bax, Bcl-2, and GAPDH antibodies were purchased from Abmart (Abmart Shanghai Co., Ltd., Shanghai, China).
9
Protein Extraction and Western Blot Analysis
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Protein extraction of whole cells was performed as previously described [24 (link)]. Equivalent levels of protein were resolved by 10%, or 15% SDS–PAGE gel (E303-01 or E305-01, Vazyme, Nanjing, China), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA), and then blocked with 1x Protein Free Rapid Blocking Buffer (Epizyme, Shanghai, China). PVDF membranes were incubated with primary antibodies overnight at 4 °C, washed with TBST three times, incubated with secondary antibodies, and last visualized by enhanced chemiluminescence using Chemiluminescence gel imaging system (Tanon 5200, Tanon, Shanghai, China). Semi-quantification of the results was assessed by Image J software (National Institutes of Health, USA). Antibodies used are as follows: MVP (1:1000, sc-23916, Santa Cruz), GAPDH (1:2000, AF2819, Beyotime), Cleaved-Caspase3 (1:1000, #9664, Cell Signaling Technology), Cleaved-Caspase8 (1:1000, #8592, Cell Signaling Technology), Bax (1:1000, #T40051, Abmart, Shanghai, China), Bcl-2 (1:1000, #T40056, Abmart), Fas (1:1000, sc-74540, Santa Cruz), ERα (1:1000, sc-8005, Santa Cruz), and Ubiquitin (1:1000, #3936, Cell Signaling Technology). Original western blots are included in Supplementary Material .
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