Ab32505

The total protein of cells or tissues was extracted using PMSF-containing RIPA lysis buffer (R0010, Solarbio, China) pre-cooled at 4 °C, followed by BCA kit (20201ES76, Yeasen, Shanghai, China) to determine the protein concentration. The proteins separated by polyacrylamide gel electrophoresis were transferred to PVDF membrane (P2438, Sigma) by wet transfer method, and the membrane was blocked with 5% BSA at room temperature for 1 h. The blots were probed with dilute primary antibody rabbit anti-KLF5 (ab137676, 1:100), AKT2 (ab32505, 1:3000), LC3B (ab51520, 1:3000, Abcam, UK), Beclin-1 (ab207612, 1:2000, Abcam, UK), and GAPDH (ab181602, 1:10000) overnight at 4 °C. Thereafter, the membrane was incubated with HRP-labeled goat anti-rabbit secondary IgG (ab6721, 1:5000) at room temperature for 1 h. The above antibodies were purchased from Abcam (Cambridge, UK). The blots were visualized using the enhanced chemiluminescence and quantified with Quantity One v4.6.2 software, with GAPDH as internal control. All blots were derived from the same experiment and were processed in parallel.

The rat hippocampal proteins were resolved on 10% SDS-PAGE gels and were subsequently transferred onto PVDF membranes (Millipore, Billerica, Massachusetts, USA). After incubation in blocking buffer (0.5% Tween-20 in TBS, 5% BSA) for 1 h at room temperature, the membranes were blotted using antibodies against the target proteins for 1 h at room temperature. Membranes were then washed with TBST (TBS with 0.5% Tween-20) and incubated in 1:1,000 or 1:5,000 diluted 0.5% BSA for 1 h at room temperature (The antibody detail: ab32575, ab32505, ab40778, ab168364, ab8226, 1:1,000, Abcam, Cambridge, MA, USA; sc-133206, 1/5000, Santa Cruz, California, USA; #13256, #2332, #12979, #9832, #2795, #2399, #12409, #15115, #2118, 1:1,000, CST, Danvers, MA, USA). After washing three times with TBST, the bands on the membrane were visualized using an ECL detection system [73 (link)].

The protein levels of HMGA2, PI3K, p‐PI3K, p‐Akt, Akt in CRC cells were measured using immunoblotting assays. Cells were lysed in RIPA lysis buffer (Cat. P0013B, Beyotime, Shanghai, China), and the protein concentration was evaluated by BCA Protein Assay Kit (Cat. P0010, Beyotime Tech.). The 20‐50 μg of protein samples were loaded onto 10% SDS‐PAGE minigel and then transferred onto a 0.2 μm PVDF membrane (Cat. 1620177, Bio‐Rad, Hercules, CA, USA) by Bio‐Rad Mini Trans‐Blot® systems (Bio‐Rad). Afterward, the membrane was probed with the antibodies listed below: anti‐HMGA2 (ab97276, Abcam, Cambridge, UK), anti‐PI3K (ab86714, Abcam), anti‐p‐PI3K (ab182651, phospho Y607, Abcam), anti‐Akt (ab32505, Abcam), anti‐p‐Akt (ab81283, phospho S473, Abcam), and anti‐β‐actin (ab6276, Abcam; 1:1000 in TBST) at 4°C overnight, and incubated with HRP‐conjugated secondary antibody for 1 hour at room temperature (1:5000 in TBST, Cat. sc‐2004 and sc‐2005, Santa Cruz, Co. Ltd, Sant Cruz, CA, USA). Signals were visualized using ECL Substrates (Cat. 345818, Millipore, Billerica, MA, USA) and exposed to X‐film (Cat. X‐OMAT, Kodak, Rochester, NY, USA). The immunoblots were quantified using imageJ software (NIH, National Institute of Health, Bethesda, MD, USA) and normalized to endogenous β‐actin. The original blots were shown in Figure S1.

Cell pellets were resuspended and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol), complemented with protease and phosphatase inhibitors cocktail (ThermoScientific). Total proteins extracts concentrations were determined through Bradford assay (Bio-Rad). Cytosol and nucleus protein fractions were obtained as previously described [47 (link)].
After 1 h blocking with 5% non-fat dried milk (EuroClone) or bovine serum albumin (SERVA) in Tris-buffered saline with 0.1% Tween (TBS-T), membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used: anti-pFGFR1 (06-1433, Millipore), anti-FGFR1 (Abcam ab137765), anti-pSTAT3 (D3A7, Cell Signaling), anti-STAT3 (06596, Millipore), anti-pAKT1 (ab81283; Abcam), anti-AKT1 (ab32505; Abcam), anti-pERK1/2 (ab32538; Abcam), anti-ERK1/2 (ab17942; Abcam), anti t-GFP (TA150041, Origene) and β-actin (Sigma, A5441). After membrane incubation with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Immuno Reagents), the positive bands were visualized using the ECL kit SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) as previously shown [48 (link)].