Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. Cells were fixed with 4% paraformaldehyde for 20 min, and then blocked with TBS (0.3% Triton X-100 and 0.25% BSA) at room temperature for 2 h. Afterwards, they were incubated overnight at 4 °C with primary antibodies: anti-PAK5 (1:100, Abcam, Shanghai, China) and β-catenin (1:100, Santa Cruz, USA) Afterwards, they were washed three-fold with PBD. Afterwards they were stained with fluorescent secondary antibodies: CoraLite488–conjugated Affinipure Goat Anti-Mouse IgG(H + L) and CoraLite594–conjugated Goat Anti-Rabbit IgG(H + L) (1:200, proteintech, China) at room temperature for 60 min. Nuclei were deal with 4′, 6-Diamidino-2-phenylindole (DAPI) (KeyGen BioTECH) for 10 min. Pictures were taken by immunofluorescence confocal laser scanning microscopy (Zeiss LSM 880).
Anti pak5
Manufactured by Abcam
Sourced in China
Anti-PAK5 is a primary antibody that recognizes the PAK5 protein. PAK5 is a member of the p21-activated kinase (PAK) family and plays a role in cell signaling pathways. This antibody can be used for applications such as western blotting, immunohistochemistry, and immunocytochemistry to detect and study the PAK5 protein.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (6 mentions)
Coralite594 conjugated goat anti rabbit igg h l, by Proteintech (2 mentions)
β catenin, by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (2 mentions)
Glass bottom culture dishes, by NEST Biotechnology (2 mentions)
Anti β catenin, by Santa Cruz Biotechnology (2 mentions)
Anti p β catenin s675, by Cell Signaling Technology (2 mentions)
Anti abcb1, by Abcam (2 mentions)
Enhanced bca protein assay kit, by Keygen Biotech (2 mentions)
β actin, by Proteintech (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Keygen Biotech (2 mentions)
Ripa lysis buffer, by Keygen Biotech (2 mentions)
Coralite488 conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Nitrocellulose blotting membrane, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose blotting membrane, by Pall Corporation (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
6 protocols using anti pak5
1
Visualizing PAK5 and β-Catenin in Cells
2
Western Blot Analysis of Apoptosis Markers
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Cell lysates were subjected to SDS-PAGE. The blots were incubated with the desired primary antibodies, which included anti-PAK5 (Abcam, ab110069), anti-cleaved caspase 3, and anti-β-catenin (both from Cell Signaling Technology, Beverly, MA), followed by incubation with an anti-rabbit IgG peroxidase-conjugated secondary antibody (Abcam, ab6721) and chemiluminescent substrates. Hybridization with anti-GAPDH (Cell Signaling Technology) was used to confirm equal protein loading.
3
Protein Extraction and Western Blot Analysis
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Cell lysis and total protein extraction were performed using the RIPA lysis buffer (KeyGen BioTECH, Jiangsu, China) and protein concentrations were gauged by Enhanced BCA Protein Assay Kit (KeyGen BioTECH). Nuclear and cytoplasmic protein was distilled via Nuclear and Cytoplasmic Protein Extraction Kit (KeyGen BioTECH). SDS-PAGE electrophoresis and nitrocellulose blotting membranes (thermo fisher scientific) were used for proteins transfer. The specific primary antibodies were incubated overnight at 4 °C, including anti-PAK5 (1:1000, Abcam, Shanghai, China), anti-ABCB1 (1:500, Abcam, Shanghai, China), anti-β-catenin (1:1000, Santa Cruz, USA), anti-p-β-catenin (S675) (1:1000, Cell Signaling Technology, USA) and β-actin (1:5000, proteintech, China). Anti-rabbit HRP or Anti-Mouse HRP (1:10,000, Vicmed) were incubated at room temperature for 2 h afterwards. Western Blot images were detected via Chemistar™ High-sig ECL Western Blot Substrate (Tanon, shanghai, China).
4
Western Blot Analysis of PAK5 Protein
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Total protein was distilled using RIPA lysis buffer (Beyotime, Shanghai, China) and thereby gauged by Enhanced BCA Protein Assay Kit (Beyotime). Equivalent proteins from each sample were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose blotting membranes (Pall Corporation, Mexico). Subsequently, the membranes were blocked with tris-buffered saline containing Tween-20 (TBST, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.05% Tween-20) and 5% non-fat dry milk for 2 h at room temperature (r/t) and incubated with specific primary antibodies: anti-PAK5 (1: 500, Abcam, Shanghai, China, catalog: ab110069) or anti-β-actin (1: 1000, ZSGB-BIO, Beijing, China, catalog: TA-09) for overnight at 4 °C. Having been rinsed thrice, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000, Vicmed, catalog: VA001 and VA002) for 2 h at r/t. Protein bands were determined using a Tanon High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) and analyzed by Image analysis software (Tanon). The grey levels of western blot analysis were measured and quantified by ImageJ software. The grey levels of PAK5 were normalized to those of β-actin and expressed as a percentage of control. All experiments were performed in triplicate.
5
PAK5 and β-catenin Colocalization Assay
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Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. Cells were xed with 4% paraformaldehyde 20 min, and then blocked with TBS (0.3% Triton X-100 and 0.25% BSA) at room temperature for 2 hours. Afterwards, they were incubated overnight at 4 °C with a primary antibody: anti-PAK5 (1:100, Abcam, Shanghai, China) and β-catenin (1:100, Santa Cruz, USA) Afterwards, they were washed three-fold with PBD. Stained with uorescent secondary antibody: CoraLite488conjugated A nipure Goat Anti-Mouse IgG(H+L) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) (1:200, proteintech, China) at room temperature for 60 min. Nuclei were deal with 4′, 6-Diamidino-2phenylindole (DAPI) (KeyGen BioTECH) for 10 min. Pictures were taken by immuno uorescence confocal laser scanning microscopy (Zeiss LSM 880).
6
Evaluating ABC Transporter Activity
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Cells lysis and total protein extraction were using the RIPA lysis buffer (KeyGen BioTECH, Jiangsu, China) and protein concentrations gauged by Enhanced BCA Protein Assay Kit (KeyGen BioTECH). Nuclear and cytoplasmic protein was distilled via Nuclear and Cytoplasmic Protein Extraction Kit (KeyGen BioTECH). SDS-PAGE electrophoresis and nitrocellulose blotting membranes (thermo sher scienti c) were used to proteins transfer. The speci c primary antibodies incubation overnight at 4 °C, including anti-PAK5
(1:1000, Abcam, Shanghai, China), anti-ABCB1 (1: 500, Abcam, Shanghai, China), anti-β-catenin (1:1000, Santa Cruz, USA), anti-p-β-catenin (S675) (1:1000, Cell Signaling Technology, USA) and β-actin (1:5000, proteintech, China). Anti-rabbit HRP or Anti-Mouse HRP (1:10,000, Vicmed) incubated at room temperature for 2 hours afterwards. The Western Blot images were detected via Chemistar™ High-sig ECL Western Blot Substrate (Tanon, shanghai, China).
Rhodamine 123 e ux assay Rhodamine 123 throughly dissolved in DMSO at a concentration of 5 mol/L. Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. 5 μM Rho123 added to the medium and incubated for 30 min at 37 °C with 5%CO 2 . Transpose new complete medium. Images were observed by immuno uorescence confocal laser scanning microscopy (Zeiss LSM 880).
(1:1000, Abcam, Shanghai, China), anti-ABCB1 (1: 500, Abcam, Shanghai, China), anti-β-catenin (1:1000, Santa Cruz, USA), anti-p-β-catenin (S675) (1:1000, Cell Signaling Technology, USA) and β-actin (1:5000, proteintech, China). Anti-rabbit HRP or Anti-Mouse HRP (1:10,000, Vicmed) incubated at room temperature for 2 hours afterwards. The Western Blot images were detected via Chemistar™ High-sig ECL Western Blot Substrate (Tanon, shanghai, China).
Rhodamine 123 e ux assay Rhodamine 123 throughly dissolved in DMSO at a concentration of 5 mol/L. Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. 5 μM Rho123 added to the medium and incubated for 30 min at 37 °C with 5%CO 2 . Transpose new complete medium. Images were observed by immuno uorescence confocal laser scanning microscopy (Zeiss LSM 880).
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