Total protein of the indicated cells was extracted using RIPA buffer (GenePharma, China), and the protein concentrations were determined using BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein were separated using 10% SDS-PAGE, and transferred to PVDF membrane (Millipore, USA). The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:2000, abcam, Cambridge, UK). Finally, protein bands were detected using ECL chemiluminescence detection kit (Beyotime, China). The primary antibodies were as following: anti-KLF12 (ab129459, 1:2000, abcam); anti-ZNF281 (ab101318, 1:2000, abcam); anti-RELA (#3033, 1:2000, Cell Signaling); anti-NRF1 (ab175932, 1:2000, abcam); anti-β-actin (ab8226, 1:2000, abcam).
Ripa buffer
Manufactured by GenePharma
Sourced in China
RIPA buffer is a commonly used protein extraction and cell lysis buffer. It is a detergent-based buffer designed to solubilize proteins from cells and tissues for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (6 mentions)
Immobilon ecl substrate, by Ipsen (2 mentions)
Amersham imager 600, by Cytiva (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (2 mentions)
Odyssey v2, by LI COR (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Anti rela, by Cell Signaling Technology (1 mentions)
Ab6721, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab175932, by Abcam (1 mentions)
Ab129459, by Abcam (1 mentions)
Ab101318, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ecl chemiluminescence detection kit, by Beyotime (1 mentions)
Sds loading buffer, by Beyotime (1 mentions)
Anti e cadherin, by Thermo Fisher Scientific (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
6 protocols using ripa buffer
1
Western Blot Analysis of Transcription Regulators
2
Protein Extraction and Western Blot Analysis
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The protein extraction was completed with RIPA buffer (GenePharma, China) with 1% General Protease Inhibitor Cocktail (MKBio, China) on ice and centrifuged at 4 °C, 12,000 rpm/15 min. The concentration of the supernatant was evaluated using the BCA Protein assay kit (Beyotime, China), and then the extracted proteins with 6 × SDS loading buffer (Beyotime, China) were denatured by heating (95 °C) for 10 min. The sample was transferred to the PVDF membrane after SDS-PAGE electrophoresis, blocked with 5% skim milk solution for 100 min, and incubated with primary antibodies at 4 °C overnight. Then, the membranes were incubated with the secondary antibody at room temperature for 1 h. Immobilon ECL substrate (EpiZyme, China) and Amersham Imager 600 (Cytiva, America) were used for signals detection and image acquisition. The antibodies used in this study were listed in Additional file 1 : Table S2.
3
Western Blot Analysis of Autophagy and Apoptosis
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Total proteins were isolated from scar fibroblasts using a RIPA buffer (Shanghai GenePharma Co., Ltd.) and were quantified with a BCA protein assay kit (Beyotime Institute of Biotechnology). Total proteins (30 µg) were separated by 10% SDS-PAGE, and then transferred onto PVDF (Bio-Rad Laboratories, Inc.) membranes. Following blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab7090; 1:5,000; Abcam) at room temperature for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to visualize the protein bands. The membranes were scanned with an Odyssey Imaging system and analyzed using the Odyssey v2.0 software (Li-Cor Biosciences). The primary antibodies used in the current study were as follows: Anti-p62 (cat. no. ab109012; 1:1,000), anti-ATG7 (cat. no. ab52472; 1:1,000), anti-Beclin 1 (cat. no. ab207612; 1:1,000), anti-Bax (cat. no. ab32503; 1:1,000), anti-Bcl-2 (cat. no. ab182858; 1:1,000), anti-cleaved caspase 3 (cat. no. ab49822; 1:1,000) and anti-β-actin (cat. no. ab8226; 1:1,000; all from Abcam). β-actin served as an internal control.
4
Proteomic Analysis of Cell Signaling
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Total protein was isolated from cell lysates using RIPA buffer (Shanghai GenePharma Co., Ltd.) and quantified by BCA protein assay kit (Beyotime Institute of Biotechnology). The proteins (40 µg per lane) were resolved on 10% SDS-PAGE and subsequently transferred onto PVDF (Bio-Rad Laboratories, Inc.) membranes. Following blocking with 5% skimmed milk in TBST (0.05% Tween-20) at room temperature for 1 h, the membranes were incubated with primary antibodies (all 1:1,000) at 4°C overnight. The following day, membranes were incubated with HRP-conjugated secondary anti-rabbit antibody (cat. no. ab7090; 1:5,000 Abcam) at room temperature for 1 h. The membranes were scanned using an Odyssey Imaging system and analyzed with Odyssey v2.0 software (LI-COR Biosciences). The primary antibodies used in the present study were as follows: Anti-β-catenin (cat. no. ab223075; Abcam), anti-SRSF1 (cat. no. 32-4500; Thermo Fisher Scientific, Inc.), anti-E-cadherin (cat. no. ab40772; Abcam), anti-Bcl-2 (cat. no. ab182858; Abcam), anti-Bax (cat. no. ab182733; Abcam) and anti-β-actin (cat. no. ab8226; Abcam). β-actin was used as an internal control. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to visualize the protein bands. ImageJ software (version 2.0; National Institutes of Health) was used to quantify the intensity of the bands.
5
Quantifying Autophagy and Apoptosis Markers
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Total proteins were obtained from lysates of cultured cells using RIPA buffer (Shanghai GenePharma Co., Ltd.). The concentration levels of the proteins were measured with a BCA protein assay kit (Beyotime Institute of Biotechnology). Subsequently, total protein (30 µg/lane) was loaded onto SDS gels (10%) and separated by electrophoresis. The gels were transferred to PVDF membranes. Following blocking with 5% skimmed milk for 1 h at room temperature, the membrane was probed with antibodies against ATG9A (1:1,000; cat. no. ab108338; Abcam), cleaved caspase-3 (1:1,000; cat. no. ab32042; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam) and beclin 1 (1:1,000; cat. no. ab210498; Abcam) overnight at 4˚C. Subsequently, the membrane was incubated with appropriate horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:3,000; cat. no. ab7090; Abcam). The visualization was performed using an ECL chemiluminescent kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The integrated density of each band was normalized to that of the corresponding β-actin (1:1,000; cat. no. ab8226; Abcam) band. ImageJ software (version 2.0; National Institutes of Health) was used to quantify the intensity of the bands.
6
Protein Extraction and Western Blot Analysis
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The protein extraction was completed with RIPA buffer (GenePharma, China) with 1% General Protease Inhibitor Cocktail (MKBio, China) on ice and centrifuged at 4°C, 12000 rpm/15 mins. The concentration of the supernatant was evaluated using the BCA Protein assay kit (Beyotime, China), and then the extracted proteins with 6x SDS loading buffer (Beyotime, China) were denatured by heating (95°C) for 10 mins. The sample was transferred to the PVDF membrane after SDS-PAGE electrophoresis, blocked with 5% skim milk solution for 100 mins, and incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with the secondary antibody at room temperature for 1 hour. Immobilon ECL substrate (EpiZyme, China) and Amersham Imager 600 (Cytiva, America) were used for signals detection and image acquisition. The antibodies used in this study were listed in Supplementary Table 2.
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