Ab193379

Western blot analysis was performed as described previously^{36 (link)}. The primary antibodies were as follows: anti-p27 (ab193379, Abcam), anti-phosphorylated Rb (Ser795) (#9301, Cell Signaling Technology), anti-E2F1 (#3742, Cell Signaling Technology), anti-p21 (#2947, Cell Signaling Technology), anti-caspase-3 antibody (ab32351, Epitomics), anti-FHL2 (ab12327, Abcam), anti-β-catenin (#8480, Cell Signaling Technology), anti-phosphorylated β-catenin (Ser33/37/Thr41) (#9561, Cell Signaling Technology), anti-cyclin D1 (60186-lg, Proteintech), anti-p53 (10442-1-AP, Proteintech), anti-PUMA (ab33906, Epitomics), anti-E-cadherin (20874-1-AP, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), and anti-GAPDH (#2118, Cell Signaling Technology).

Western blot analysis was performed as described previously^{40 (link)}. The following primary antibodies were used: anti-cyclin D1 (60186-lg, Proteintech, Chicago, IL, USA), anti-p27 (ab193379, Abcam, Cambridge, UK), anti-p21 (#2947, Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated Rb (Ser795) (#9301, Cell Signaling Technology), anti-IRS1 (#2382, Cell Signaling Technology), anti-phosphorylated IRS1 (Ser307) (D151214, BBI, Shanghai, China), anti-IGF1R (20254-1-AP, Proteintech), anti-phosphorylated AKT (Ser473) (66444-1-lg, Proteintech), anti-AKT (10176-2-AP, Proteintech), anti-phosphorylated mTOR (Ser2448) (D155324, BBI), anti-mTOR (20657-1-AP, Proteintech), anti-FoxP3 (D260367, BBI), anti-Dicer (ab14601, Abcam), anti-Drosha (55001-1-AP, Proteintech), and anti-GAPDH (#2118, Cell Signaling Technology).

RIPA lysis buffer was used to extract protein, and the protein concentration was quantified using the BCA protein concentration determination kit. The proteins (50 μg per sample) were resolved by electrophoresis under 100 V constant pressure and subsequently electro-transferred onto a PVDF transfer membrane. Then, the membrane was incubated in 5% bovine serum albumin (BSA) in TBST buffer at room temperature for 2 h, followed by incubation at 4 °C overnight with the primary antibodies as follows: anti-β-actin (1:5000; ab179467, Abcam), anti-CDC2 (1:1000; ab201008, Abcam), anti-CDK2 (1:1000; ab32147, Abcam), anti-Cyclin B1 (1:2000; ab181593, Abcam), anti-P21 (1:1000; ab188224, Abcam), anti-P27 (1:2000; ab193379, Abcam), anti-Wnt1 (1:500; 27935-1-AP, Proteintech), anti-β-catenin (1:500; ab68183, Abcam), anti-PRC1 (1:5000; ab51248, Abcam), and anti-NUF2 (1:1000; ab230313, Abcam). All antibodies were diluted with a TBST buffer containing 5% BSA. Next, the membranes were washed three times with TBST and incubated with secondary antibodies for 1 h. The membranes were then incubated with ECL chemiluminescence, and the bands were observed using a UVP chemiluminescence imaging system. We used β-actin protein as the internal reference protein to calculate the expression of the protein to be tested.

Rabbit monoclonal antibody (Ab) against Bax (#2772) and rabbit polyclonal Ab against Bak (#3814) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal Ab against p27 ^Kip1 (ab193379), rabbit monoclonal Abs against p16^INK4 (ab51243), p21^Cip1 (ab109199), p57 ^Kip2 (ab75974), and β-actin–horseradish peroxidase (HRP) (ab49900) were purchased from Abcam (Cambridge, UK). Alexa fluor secondary Abs were purchased from Thermo Fisher. HRP Abs were purchased from Southern Biotechnologies Associates (Birmingham, UK).

Human tubular epithelial cells HK‐2 were purchased from the America Tissue Culture Collection. The reverse transcription and SYBR Green qRT‐PCR kits were purchased from TOYOBO, Japan.
The following antibodies were used in this study: rabbit anti‐caspase3 antibody (1:1,000, 8G10, 9665; Cell Signaling Technology), rabbit anti‐cleaved‐PARP antibody (Asp214) (1:1,000, D6X6X, 94885, Cell Signaling Technology), rabbit antibody to beclin‐1 antibody (1:1,000, D40C5, 3495, Cell Signaling Technology), rabbit anti‐LC3B antibody (1:2,000, EPR18709, ab192890, Abcam), rabbit anti‐DCHS1 antibody (1:1,000, Ab203690; Abcam), rabbit anti‐cyclin D1 antibody (1:200, SP4, ab16663, Abcam), rabbit anti‐CDK4 antibody (1:2,000, EPR17525, ab199728, Abcam), mouse anti‐p27 antibody (1 μg/ml, SX53G8, ab193379, Abcam), and rabbit anti‐LC3B antibody (1 μg/ml, EPR18709, ab192890, Abcam).

Antibody against PHLDA1 was obtained from Invitrogen (#PA5-53889), antibodies against actin (#ab109193), phosphor-AKT (#ab81283), AKT (#ab182729), p27 (#ab193379), p53 (#ab26), bax (#ab32503), bcl-2 (#ab59348) were obtained from Abcam. All primers for Q-PCR were ordered from Hongxun (Suzhou, China).