β actin ap0060

Total protein was extracted from the EC cell lines using a radioimmunoprecipitation assay (RIPA) kit (Solarbio). The protein samples were separated by 10% SDS–PAGE (Sangon Biotech, Shanghai, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, USA) according to a standard process. After blocking in 5% skim milk (BD Difco, Maryland, USA) or 5% bovine serum albumin(BSA, Biofroxx, Einhausen, Germany) for 2 h, the membranes were incubated overnight at 4 ^oC with primary antibodies against DCLK1 (JA11-03, HUABIO, Hangzhou, China), E-cadherin (ab40772, Abcam, Cambridge, UK), N-cadherin (ab76011, Abcam), Cyclin D1 (26,939-1-AP, Proteintech, Wuhan, China), CDK4 (#12,790, Cell Signaling Technology, Massachusetts, USA), PI3K p85 (ab191606, Abcam), p-PI3K p85 (phospho Y607) (ab182651, Abcam), Akt (60,203-2-lg, Proteintech), p-Akt (phospho Ser 473) (66,444-1-lg, Proteintech), NF-κB p65 (ab76311, Abcam), or NF-κB p-p65 (phospho S536) (ab76302, Abcam), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies(LF101 or LF102, Epizyme, Shanghai, China) for 2 h (1:5000). β-actin (AP0060, Bioworld, Nanjing, China) or GAPDH (AP0063, Bioworld) antibodies were used as the internal controls at a dilution of 1:5000. The protein signals were analyzed using the enhanced chemiluminescence method (Millipore).

Total proteins were extracted from islets, and protein levels were measured by the BCA method. Equal quantities of protein were electrophoresed using a TGX Stain-Free FastCast Acrylamide Kit, 10% (BIO-RAD) and then transferred onto PVDF membranes (Millipore). The membranes were incubated in TBST buffer containing 5% BSA (Boster Biological Technology Co., Ltd, Wuhan, China) to block non-specific binding at room temperature and then were incubated with primary antibodies at 4°C overnight. On the next day, the membranes were washed in TBST buffer and then were incubated with secondary antibodies for 1 h at room temperature. Finally, the proteins bands were visualized using ECL Plus and the ChemiDocTM Imaging System (BIO-RAD). The band intensities were analyzed with ImageJ software (US National Institutes of Health). Protein levels were normalized to the β-actin protein, and phosphorylated forms were normalized to phosphorylation-independent levels of the same protein. Antibodies used in this study are listed in Table 2.
Table 2

Antibodies used for Western blot analysis.

Antibody	Catalog no.	Company	Dilution ratio
TXNIP	ab188865	Abcam	1:2000
Cyclin D2	ab230883	Abcam	1:2000
CDK4	ab137675	Abcam	1:3000
p27	ab92741	Abcam	1:5000
p-PI3K	17366s	CST	1:1000
PI3K	4292s	CST	1:1000
p-AKT	9271s	CST	1:1000
AKT	9272s	CST	1:1000
p-mTOR	5536s	CST	1:1000
mTOR	2983s	CST	1:1000
p-GSK3β	5558s	CST	1:1000
GSK3β	9315s	CST	1:1000
β-actin	AP0060	Bioworld	1:10,000