Gapdh

Mutant and wild-type WEE2 plasmids were transfected into HeLa cells with Lipofectamine 3000 Reagent according to the manufacturer’s instructions (L3000008; Invitrogen). After 36 h of incubation, transfected cells were lysed and subjected to SDS-PAGE. Primary antibodies against FLAG (8146, dilution: 1:1000; CST) and pY15-Cdc2 (4539, dilution: 1:1000; CST) were used to detect the mutation effects of pDRF-FLAG-WEE2 plasmids. Primary antibodies against GAPDH (60,004, dilution: 1:20,000; Proteintech, Wuhan, China) and Cdc2 (77,055, dilution: 1:1000; CST) were used as internal controls. The secondary antibodies, horseradish peroxidase-linked anti-rabbit and anti-mouse IgG (7074 and 7076, dilution: 1:2000; CST), were incubated with the membranes. Finally, bound proteins were visualized by enhanced chemiluminescence (WBKLS0500; Millipore Corp., Billerica, MA, USA).
To determine the effects of WEE2 mutant variants on subcellular localization, compartment extraction of transfected HeLa cells was conducted with the Nuclear and Cytoplasmic Extraction Reagents Kit (78,833, Thermo Scientific). The nuclear and cytoplasmic compartments were quantified by western blotting using antibodies against Lamin B1 (66,095–1-lg, dilution: 1:10,000; Proteintech) and GAPDH (60,004, dilution: 1:20,000; Proteintech) to normalize the quantities of nuclear and cytoplasmic proteins, respectively.

Western blot and RT-qPCR were performed as previously described [23 (link)].
The following primary antibodies were used: APP, amyloid-β precursor protein (Proteintech, 25524-1-AP, 1:500 dilution), GAPDH (Proteintech, 60004-1-Ig, 1:20000 dilution).
The primer sequences were as follows: GAPDH, F: 5′-AGAAGGCTGGGGCTCATTTG-3′; R: 5′-AGGGGCCATCCACAGTCTTC-3′; APP, F: 5′-TCTCGTTCCTGACAAGTGCAA-3′; R: 5′-GCAAGTTGGTACTCTTCTCACTG-3′.

The cells were lysed on ice with the RIPA (Radio Immunoprecipitation Assay) lysis buffer (Thermo Fisher Scientific, USA) for 30 min to prepare the cell suspension, followed by centrifugation at 14,000 rpm for 10 min at 4 °C. The protein concentration was determined with bicinchoninic acid protein assay (Thermo Fisher Scientific, USA), 0.1 mg total protein were subjected to 10% SDS-PAGE separation and then transblotted to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked in 5% nonfat milk for 1 h at room temperature, and then incubated with the primary antibodies. Target proteins were detected by using specific antibody against CALM1 (catalog number: #10,541–1-AP; dilution at 1:800; Proteintech Group, Wuhan, China). GAPDH (catalog number: 10494–1-AP; dilution at1:5000, Proteintech Group, Wuhan, China) was chosen as an internal control and the CALM1 and GAPDH dilutions were incubated at 4 °C with gentle shaking overnight. Then, secondary antibody (goat anti-rabbit, catalog number:SA00002-2, Proteintech Group, Wuhan, China) were added onto the membrane for incubation at room temperature for 2 h.The blots were visualized with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, CA, USA), according to the manufacturer's protocol.

HCC cells were lysed with RIPA buffer (Beyotime) containing protease inhibitors and/or phosphatase inhibitors (Beyotime) for 20 min on ice. The lysate was centrifuged at 12000 g for 10 min at 4°C, and the supernatant was collected. The concentration of the supernatant was measured by a BCA Protein Assay Kit (Beyotime). After mixing with loading buffer, the proteins were denatured at 100°C in a metal bath for 10 min. The proteins were separated and then transferred to PVDF membranes (Millipore). After blocking with 5% skimmed milk for 2 h, the membrane was incubated with antibodies (pAkt: 1:2000, Proteintech; GAPDH: 1:10000, Proteintech) overnight at 4°C. The PVDF membrane was blocked with 5% skimmed milk for 2 h at room temperature and incubated with antibodies (pAkt: 1:2000, Proteintech; GAPDH: 1:10000, Proteintech) overnight at 4°C. After washing with TBS, the membranes were incubated with a homologous HRP‐conjugated secondary antibody at room temperature for 1 h. Finally, the membranes were detected using a gel imaging system (Vilber, France) with Clarity Western ECL Substrate (Bio‐Rad).

The western blot analysis procedure was conducted as previously reported¹¹. The primary or secondary antibodies and their respective diluted concentrations we used in this study are as follows: (LHPP: dilution 1:200, catalog no. 15759‑1‑AP, Proteintech; GAPDH, dilution 1:2000, catalog no. 60004‑1‑AP, Proteintech; β-actin, dilution 1:2000, catalog no. 20536‑1‑AP, Proteintech; AKT, dilution 1:500, catalog no. 10176‑2‑AP, Proteintech; p‑AKT, dilution 1:1000, catalog no. 4060S, CST; PI3K, dilution 1:1000, catalog no. 4249S, CST; p-PI3K, dilution 1:1000, catalog no. ab278545, abcam; E-cadherin, dilution 1:1000, catalog no. 9782T, CST; β-catenin, dilution 1:1000, catalog no. 9782T, CST; N-cadherin, dilution 1:1000, catalog no. 9782T, CST; Vimentin, dilution 1:1000, catalog no. 9782T, CST; Snail , dilution 1:1000, catalog no. 9782T, CST; Slug, dilution 1:1000, catalog no. 9782T, CST; ZEB1, dilution 1:1000, catalog no. 9782T, CST; mTOR, dilution 1:1000, catalog no. 9862T, CST; p-mTOR, dilution 1:1000, catalog no. 9862T, CST; p-pS6k(C371), dilution 1:1000, catalog no. 9862T, CST; p-pS6k(T389), dilution 1:1000, catalog no. 9862T, CST; 1-Histidine phosphorylation (1- PHis), dilution 1:1000, catalog no. MABS1330, Merk; 3-PHis, dilution 1:1000, catalog no. MABS1352, Merk); secondary antibody (dilution 1:10000, catalog no. SA00001-2/ SA00001-1, Proteintech).