Anti pi3k

Baicalin (purity ≥ 98%) was obtained from Xi’an Kai Lai Biological Engineering Co., Ltd. (Xi’an, China). Fluoxetine hydrochloride (Flu) was purchased from Changzhou Siyao Pharmaceuticals Co., Ltd. (Changzhou, China). Lipopolysaccharide (LPS), 4,6-diamidino-2-phenylindole (DAPI), and compound C were bought from Sigma-Aldrich Co (St. Louis, USA). TAK-242 (a TLR4 antagonist) and LY294002 (a PI3K inhibitor) were products purchased from Apex Bio (Houston, USA). Poly-d-lysine was obtained from Sigma (MO, USA). Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were supplied by Elabscience Biotechnology Co., Ltd. (Wuhan, China). The antibodies were obtained from the cited commercial sources: anti-p-PI3K (#4228), anti-PI3K (#4292), anti-p-Akt (Ser473, #9271), anti-Akt (#9272), anti-β-actin (#4967), anti-PCNA (#13110), anti-p-AMPK (#2531), and anti-AMPK (#2603) were from Cell Signaling Technology (Beverly, MA, USA); anti-PTEN (sc-7974) and anti-p-PTEN (sc-377573) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-TLR4 (AF7017), anti-p-GSK3β (AF2016), and anti-GSK3β (AF7814) were from Affinity Biosciences (Changzhou, China); and anti-FoxO1 (ab52857), anti-p-FoxO1 (Ser 256, ab131339), and goat anti-rabbit IgG H&L (Alexa Fluor® 488, ab150077) were from Abcam (Cambridge, MA, USA).

After washing with PBS, the cells were lysed with prechilled lysis buffer. The collected cell lysate was centrifuged at 14000 × g for 15 min at 4°C, and the protein content was measured with 5 × sample buffer (BSA; Thermo Fisher Scientific, Inc., CA, USA). Then, these protein samples were tested by WB. The 4%-20% precast gel was used to transfer the protein to the nitrocellulose membrane, and the 5% skimmed milk was used to seal the membrane at ambient temperature (Bio-Rad Laboratories) 1 h. Subsequently, specific antibodies (diluted 1 : 10 00), including anti-FGF12 (ab231956, Abcam), anti-E-cadherin (ab231303, Abcam), anti-Vimentin (ab8978, Abcam), anti-p-PI3K(#17366, Cell Signaling Technology), anti-PI3K(#4249, Cell Signaling Technology), anti-p-AKT(S473)(#4060, Cell Signaling Technology), anti-N-cadherin(ab18203, Abcam), anti-p-AKT(T308)(#13038, Cell Signaling Technology), anti-AKT(#4685, Cell Signaling Technology), and anti-β-actin (#4970, Cell Signaling Technology), were used to block membranes. Subsequently, the secondary antibody was used to incubate the membranes (Santa Cruz) at ambient temperature for 1 h and visualized them using ECL solution (Bio-Rad Laboratories). Finally, a chemiluminescence imaging system (Mini HD9; UVitec, Cambridge, UK) was used to take images.

Total proteins were extracted from cells or tissues using RIPA Lysis Buffer. Protein concentration was measured by BCA Protein Assay Kit. To examine the expression of proteins, the same amount of total proteins was loaded on an 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were transferred into PVDF membranes. After being blocked in skim milk solution, the membrane was incubated overnight separately with antibodies anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PI3K, anti-p-Akt, anti-p-Bad, anti-Cyt-c, anti-cleaved caspase 9, anti-cleaved caspase 3, anti-PARP, anti-DRP1, and anti-Mfn2 (Cell Signal, CST, USA). After that, the membrane was incubated with the secondary HRP-conjugated goat anti-rabbit antibodies (Santa Cruz Biotechnology). Proteins were visualized using an enhanced chemiluminescence kit from Thermo Fisher Scientific (Massachusetts, USA). ImageJ software (Alpha View SA) was used to perform densitometric analysis.

Thirty μg of protein was separated by 12% SDS-PAGE and transferred onto a 0.22-μm PVDF membrane (Millipore, Boston, MA, USA). The following primary antibodies were used to incubated the membrane at 4°C overnight: anti-Bcl2L12 (1:500, Abcam, Cambridge, UK), anti-EGFR (1:500, Abcam), anti-PI3K (1:400, Cell Signaling Technology (CST), Boston, MA, USA), anti-p-PI3K (1:200, CST), anti-caspase-7 (1:300, Abcam), and anti-β-actin (1:800, Abcam) that was used as the internal reference. After washing and incubating with the corresponding HRP-conjugate secondary antibodies, the membrane was analyzed with the Enhanced Chemiluminescent (ECL) Western blotting Kit (Millipore) in a Gel Imaging System (Bio-Rad).

Proteins were electrophoresed by SDS/PAGE (12% or 10% gel) and the blots were incubated overnight with primary antibody. The following primary antibodies were used: anti-ALOX12 (Santa-Cruz, sc-365,194), anti-E-Cadherin (Cell signal Technology, #14472), anti-N-Cadherin (Cell signal Technology, #13116), anti-Vimentin (Cell signal Technology, #5741), anti-Snail (Cell signal Technology, #3879), anti-Slug (Cell signal Technology, #9585), anti-MMP2 (Abcam, ab37150), anti-MMP7 (Abcam, ab5706), anti-MMP9 (Abcam, ab38898), anti-MMP13 (Abcam, ab39012), anti-GPR31 (Abcam, ab75579), anti-PI3K (Cell signal Technology, #4249), anti-AKT (Abcam, ab179463), anti-AKT (phospho T308, Abcam, ab38449), anti-NF-κB (Abcam, ab16502), anti-NF-κB (phospho S536, Abcam, ab86299), anti-GAPDH (Abcam, ab181602).

All the patients signed the written informed consents before enrollment, and the procedures were approved by the ethical committees of The First Affiliated Hospital of Shandong First Medical University (No. 2017-S007). And 3 cases of ccRCC tissues (including cancer and adjacent non-cancerous), which were collected from 3 patients (2 males and 1 females, age from 54 to 65, Fuhrman nuclear grading with 1 G2 + 2 G3, TNM staging with 2 T1 + 1 T2) (Edge & Compton, 2010 (link)), were used for WB analysis as described previously (Zhao et al., 2017 (link)). After incubated with the corresponding primary antibodies: anti-PI3K (1:1,000, rabbit, #4249; Cell signaling Technology, Danvers, MA), anti-AKT (1:1,000, rabbit, #4691; Cell signaling Technology, Danvers, MA), anti-mTOR (1:2,000, rabbit, ab2732; Abcam, Cambridge, MA), and anti-β-Actin (1:1,000, mouse, BM0627; BOSTER, Beijing, China), horseradish peroxidase (HRP) conjugated secondary antibodies (1:5,000, rabbit, SA00001-2 & mouse, SA00001-1; Proteintech, Wuhan, China) were used to detect the proteins. Then the blots were visualized by enhanced chemiluminescence (Amersham Imager 600, GE Healthcare, Marlborough, MA). Protein ladder (MAN0011772, Thermo Scintific, Waltham, MA) was used to label the corresponding proteins, and Image J software was used for quantification.