Anti pi3k

Collected whole cellular proteins and then determined the protein concentrations by BCA. Next, 45 μg proteins was resolved over 10% polyacrylamide gels and then transfer-embedded onto a PVDF membrane (Solarbio, Beijing, China). Blocking of the membrane was done in 5% non-fat milk for two hours at RT and then overnight inoculated with the appropriate primary antibodies at 4° C. For PI3K/AKT/mTOR pathway, anti-PI3K (ABclonal; 1:1000), anti-AKT (ABclonal; 1:1000), anti-p-AKT (Abcam; 1:1000), anti-mTOR (Abcam; 1:1000) and anti-p-mTOR (Abcam; 1:1000) antibodies were used. For cellular apoptosis, anti-c-PARP (1:1000, Proteintech), anti-caspase-3 (Proteintech, Wuhan, China; 1:1000) and anti-caspase-8 (Proteintech; 1:1000) antibodies were used. For EMT-related proteins, anti-N-cadherin (Abcam; 1:1000), anti-E-cadherin (ProteinTech; 1:1000), anti-α-SMA (Affinity Biosciences; 1:1000), anti-Vimentin (Affinity Biosciences; 1:1000), anti-Collagen I (Abcam; 1:1000) and anti-Collagen III (Abcam; 1:1000) were used. GADPH (Bioworld; 1:1000) was employed as the internal reference. After rinsing thrice in TBST, incubated the membranes with anti-rabbit secondary antibody (Proteintech; 1:5000) for one hour at RT. Lastly, the protein bands were rinsed five times in TBST and visualization via chemiluminescence detection done on the autoradiographic film.

Cell protein was extracted using a lysis buffer (P0013B, Beyotime, Nanjing, China), which was supplemented with protease and phosphatase inhibitor cocktail (ST506, Beyotime, Nanjing, China; GB-0032, KeyGEN, Nanjing, China). After incubation on ice for 30 min, the lysates were centrifuged at 12,000 rpm for 10 min at 4 ℃ and the supernatants were collected. The total proteins were separated by 6%-10% SDS-PAGE gels, then immediately transferred onto PVDF films (Millipore, Darmstadt, Germany). The films were furtherly probed by primary antibodies overnight (4℃), and then incubated with corresponding secondary antibodies. A BIORAD imaging system (chemiDOCTMXRS, USA) was applied to visualized the protein bands. The primary antibodies used in this study were listed as follows: Anti-TRPV1 (NB100-1617, Novus Biologicals, Colorado, USA), Anti-SR-3A (sc-390168, Santa Cruz, USA), Anti-5HT-H209 (NB120-16007, Novus Biologicals, Colorado, USA), anti-mTOR (2983, CST, Boston, USA), anti-p-mTOR (5536, CST), anti-PI3K (A0265, ABclonal, Wuhan, China) and anti-GAPDH (A19056, ABclonal, Wuhan, China).