Following the respective treatments, the cells were harvested and lysed in RIPA buffer (Cell Signaling, Danvers, MA, USA). The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were separated on 10% SDS‐polyacrylamide gels. The proteins were transferred to a PVDF membrane and probed with antibodies targeting CLIC1 (Abcam, UK; 1:1000 dilution), PI3K 110α (Cell Signaling Technology, USA; 1:1000 dilution), p‐Akt (Cell Signaling Technology, USA; 1:1000 dilution), Akt (Cell Signaling Technology, USA; 1:1000 dilution), Bcl‐2 (Abways, China; 1:1000 dilution), Bax (Abways, China; 1:1000 dilution) and β‐actin (Abways, China; 1:1000 dilution). The blots were then incubated with HRP‐conjugated secondary antibodies followed by enhanced chemiluminescence (ECL) detection.
Bcl2 associated x (bax)
Manufactured by Abways
Sourced in China
Bax is a precision laboratory instrument designed for accurate measurement and analysis. It features advanced sensors and a user-friendly interface to provide reliable data. The core function of Bax is to perform precise quantitative assessments within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Abways (4 mentions)
P akt, by Cell Signaling Technology (2 mentions)
β actin, by Abways (2 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Clic1, by Abcam (1 mentions)
Pi3k 110α, by Cell Signaling Technology (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Cytochrome c, by Abways (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Vimentin, by Abways (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
E cadherin, by Abways (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Phospho p70s6k thr389, by Cell Signaling Technology (1 mentions)
Ecl solution, by Applygen (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
5 protocols using bcl2 associated x (bax)
1
Western Blot Analysis of Protein Expression
2
Chloroquine-Mediated Protein Extraction
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Cells are treated with chloroquine for 12-24 hours, the cell pellet is collected by centrifugation, add lysis buffer (Beyotime Biotechnology, Shanghai, China), and protein is collected. After the animal experiment treatment is over, the subcutaneous tumor is taken out, the tissue is frozen and ground with liquid nitrogen, the lysate is added, and the tissue protein is extracted. Use the BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China) to determine the protein concentration and determine the sample volume. After the electroporation is over, 5% skim milk is blocked at room temperature for 1-2 hours, and the primary antibody is incubated at 4°C overnight. The primary antibodies are as follows: PD-1(1:1000, Abcam, MA), MMP2,Cleaved-caspase-3,PD-L1,CD4,CD8 (1:1000,CST,USA), Bax, Bcl-2, Cytochrome-C(1:1000, Abways, China), and Tubulin (1:3000,Sigma,USA), After incubating for 1 hour with the secondary antibody corresponding to the primary antibody, wash the PVDF membrane (Millpore, USA) and use ECL chemiluminescent solution (Millpore, USA) for chemical imaging. Use Image J software for gray value analysis.
3
Chaetoglobosin E Modulates Cell Signaling
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KYSE-30 cells were seeded in six-well plates (3×105 cells/well). After 24 h, the cells were treated with different concentrations of chaetoglobosin E for 48 h, and then collected and washed with PBS. The cellular proteins were extracted using a protein extraction kit (Keygen, China). The proteins were separated in 10% sodiumdodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gels according to their different molecular weights, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by wet rotation. The PVDF membranes were then blocked with 5% skim milk for 1 h and incubated with primary antibodies to ERK, p-ERK, p-MEK, Akt, p-Akt (Cell Signaling Technology, America), PLK1 (Abcam, UK), MEK, EGFR, p-EGFR, GSDME, Bcl-2, Bax, beclin1, LC3, p21, cyclinB, CDC2, p-CDC2, E-cadherin, vimentin, and Actin (Abways, China) at 4 ℃ overnight. The following day, the PVDF membranes were washed with tris buffered saline-Tween-20 (TBST) and incubated with the appropriate secondary antibody for 1 h. The protein bands were imaged using the BLT GelView 6000Plus (Guangzhou, China).
4
Protein Expression Analysis in Lung Tissue
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Lung tissues and cells were lysed conventionally using the RIPA lysis buffer containing a mixture of PMSF and phosphatase inhibitor (v/v = 97:1:2) separately on ice. The total protein concentration was determined by bicinchoninic acid (BCA) protein assay. Equal amount of proteins was subjected to 10% SDS/PAGE and transferred on to polyvinylidene fluoride (PVDF) membranes [16 (link)]. After the transfer, the blots were blocked with 5% skimmed milk for 2 h and incubated overnight with diluted primary antibody B-cell lymphoma 2 (Bcl-2; Wanleibio, 1:500), Bax (Abways, 1:1000), β-actin (Abways, China, 1:3000), protein kinase B (Akt/PKB; Wanleibio, 1:500), phospho-Akt (Ser473, Wanleibio, 1:500), phospho-mTOR (Ser2448, Wanleibio, 1:500), mTOR (Wanleibio, 1:500), phospho-p70S6k (Thr389, Cell Signaling Technology, 1:500), p70S6k (Cell Signaling Technology, 1:1000). After washing with PBS (10 min × 3-times), bands were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Abways, China, 1:5000) for 2 h at room temperature. The blots were then developed using enhanced chemiluminescence (ECL) solution (Applygen Technologies Inc.). The semi-quantitative analysis was performed using densitometry analysis by ImageJ 1.50i.
5
Protein Expression Analysis of DLL4/Notch Pathway
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After treatment for 72 h, the cells were lysed and total proteins were extracted using RIPA buffer with protease inhibitors (Beyotime Biotechnology). Then, the protein concentration was determined by the BCA assay kit (Beyotime Biotechnology). Equal amounts of protein samples (40 μg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membranes were blocked in 5% fat-free milk, then cut according to the size of the target protein and incubated with primary antibodies against DLL4 (1:1000, Abcam), Notch (1:1000, Abcam), Hes1 (1:1000; Abcam), Bax (1:1000, Abways Technology), Bcl-2 (1:1000, Abways Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology) and GAPDH (1:1000) (Beyotime Biotechnology) at 4 °C overnight and then incubated with secondary antibodies (1:4000, Beyotime Biotechnology) at room temperature for 1 h39 (link). Finally, the bands were exposed by ECL (Tanon, Shanghai, China).
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