Ab180710

The cells were harvested, and total protein was extracted using RIPA lysis buffer. The protein samples (30-50 μg) were loaded on an SDS-gel, resolved using SDS-PAGE, and transferred to a PVDF membrane (EMD Millipore). The membranes were blocked for 1 h with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween (TBS-T) and then incubated with the one of the following primary antibodies: anti-FOXM1 (1 : 500; Abcam, ab180710), GAPDH (1 : 500; Abcam, ab8245), or anti-ABCC5 (1 : 500; Abcam, ab180710) at 4°C overnight. Membranes were subsequently washed in TBS T three times and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1 : 2,000; Santa Cruz Biotechnology, Inc.). Signals were visualized using a Pierce ECL Western Blotting Substrate detection system (Thermo Fisher Scientific, Inc.).

Double immunofluorescence staining was implemented as established protocols.¹⁰ The mouse kidney sections were incubated with anti‐FoxM1 (ab1807.10; Abcam) and anti‐AQP1 (ab9566; Abcam). NRK‐52E cells were incubated with adenovirus expressing rat FoxM1 gene (Ad‐FoxM1) or empty vector (Ad‐null), then followed by fixation at 37°C using 4% paraformaldehyde for 20 minutes. Fixed cells were hybridized with primary antibodies against FoxM1 (sc‐271746; Santa Cruz) and active β‐catenin (05‐665; Millipore) at 4°C overnight. Then, secondary antibodies (Proteintech) and 4′,6‐Diamidino‐2‐phenylindole (DAPI; Beyotime) visualized primary antibodies and nuclei, respectively. Cells were viewed using a confocal biological microscope (ZEISS).

The total protein was extracted from cultured melanoma cells using the RIPA buffer (Beyotime, Shanghai, China) to detect the expression level of CCT3. The protein concentration of each sample was determined using the BCA protein assay kit (Blue Skies, Shanghai, China). 10% SDS-PAGE gel electrophoresis was used for separating total proteins, then the proteins were transferred onto polyvinylidene difluoride membranes. After the membranes were blocked with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies: mouse anti-GAPDH (1:2000, Santa Cruz, sc-2005), mouse anti-β-actin (1:10000, Abcam,ab6276), rabbit anti-CDK1 (1:300, Abcam, ab32094), rabbit anti-FOXM1 (1:400, Abcam, ab180710), rabbit anti-MCM-2 (1:1000, Abcam, ab109271), rabbit anti-NFKBIA (1:300, Abcam, ab7217), rabbit anti-PIM1 (1:100, Abcam, ab75776), and rabbit anti-SKP2 (1:300, Abcam, ab183039), rabbit anti-Bcl-2 (1:500, Abcam, ab692), rabbit anti-Bax (1:500, Abcam, ab32503), and rabbit anti-Cleaved PARP (1:1000, Abcam, ab32064). The membranes were then washed with TBST 3 times and incubated with the secondary antibody (Yeasen, Shanghai, China) for 2 h at room temperature. Bands were visualized using a chemiluminescent HRP substrate (Millipore, MA, USA) and an electrogenerated chemiluminescence imaging system (Tanon, Shanghai, China) 26 .