Pthr1

Whole-cell protein was extracted with the RIPA lysis buffer containing protease inhibitor (Beyotime) and protein phosphatase inhibitor (Beyotime) on ice. Equal amounts of protein (30 μg) were loaded into 10% (w/v) SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, St. Louis, MO, USA), with the membrane then incubated with primary antibodies at 4 °C overnight, followed by three series of TBST washing and 2 h of HRP-conjugated secondary antibodies incubation at room temperature. The proteins were visualized through chemiluminescence and imaged on a Gelview 6000 Pro (BLT Co., Ltd, Guangzhou, China). The protein bands were quantified by densitometry analysis using Image J software.
The primary antibodies used in this study included antibodies against GAPDH (1:1000; Affinity Biosciences), Vim (1:1000; Abcam), E-cad (1:1000; Affinity Biosciences), N-cad (1:1000; Santa Cruz, Cincinnati, OH, USA), PI3K (1:1000; Santa Cruz), AKT (1:1000; Santa Cruz), p-PI3K (1:1000; Affinity Biosciences), p-AKT (1:1000; Affinity Biosciences), and PTHR1 (1:1000; Affinity Biosciences).

HaCaT cells were seeded (10⁴ cells/mL) in confocal dishes and different treatments were applied for 48 h. Then, the HaCaT cells were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilizing with 0.5% (v/v) Triton X-100 (Solarbio, Beijing, China) for 15 min. After washing with PBS three times, the cells were blocked in 5% bovine serum albumin (BSA, Solarbio) for 1 h and incubated in primary antibodies overnight at 4 ℃. Subsequently, the cells were washed again and incubated in Alexa Fluor 488- or 594-conjugated secondary antibodies (Bioss, Beijing, China) for 2 h at room temperature. Finally, 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) was utilized to stain the nuclei for 10 min. Fluorescence images were observed by confocal laser scanning microscopy (LSM-980; Carl Zeiss, Oberkochen, Germany). The primary antibodies used in this study included primary antibodies against Ki67 (1:1000; Affinity Biosciences, Cincinnati, OH, USA), Vim (1:1000; Abcam, Cambridge, UK), E-cad (1:1000; Affinity Biosciences), p-AKT (1:1000; Affinity Biosciences), and PTHR1 (1:1000; Affinity Biosciences).