P akt 9271

All phospho-antibodies (pIGF1R #3024, pEGFR #2234, pAKT #9271, pMAPK #9101) and polyclonal antibodies to AKT (#9272) and IGF1R (#3027) were from Cell Signaling Technology (Beverly, MA). Monoclonal mouse antibodies to SALL4 (#sc-101147) and CBL-B (#sc-1705), and polyclonal antibody to EGFR (#sc-03) were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody to MAPK (#61-7400) was from Invitrogen Life Technologies (Carlsbad, CA). HDAC inhibitors (trichostatin A and entinostat) were provided by Dr. James Bradner's lab at DFCI.

Frozen spleen tissue was ground with mortar and pestle on dry ice, resuspended in cold RIPA buffer (Sigma-Aldrich, St Louis, MO, USA) containing a protease inhibitor and phosphatase inhibitor cocktail and then homogenized. The protein supernatant was collected and the protein concentration was determined using a bicinchoninic acid protein assay kit (BCA assay, Pierce, Rockford, IL, USA). The proteins were diluted in NuPAGE LDS Sample Buffer (4×) and denatured at 95 °C for 10 min. Following this, 50 µg of protein (in total 30 µL volume) was loaded per lane in a 4–12% Bis-Tris 18-well Criterion XT precast polyacrylamide gel (BioRad, Hercules, CA, USA). The gel was run at 70 V on the Criterion Midi format Cell™ (BioRad), then transferred using Trans-blot Turbo transfer system onto PVDF membrane. Blocking and incubation of membranes with primary and secondary antibodies were carried out using the Starting Block (ThermoFisher). Blots were washed using 0.05% TBST and TBS. The following antibodies were used: β-Actin (8457), JNK (9252), p-JNK (4668), AKT (9272), and pAKT (9271) from Cell Signaling. β-actin was used as the protein loading control. Densitometric analyses of protein bands were performed using Image Lab 5.2.1 Software from BioRad [33 (link)].

After exposure or not to LIPUS, HaCaT cells were lysed in RIPA buffer (Beyotime, China) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF; Beyotime). The whole lysates were centrifuged at 12,000 g for 10 min at 4°C, and the protein levels in the supernatants were quantified using the BCA™ Protein Assay Kit (Pierce, USA). Proteins were loaded onto SDS-PAGE gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Then, PVDF membranes were blocked with 5% bovine serum albumin (BSA; Solarbio, China), and incubated overnight at 4°C with specific primary antibodies for cyclin D1 (ab134175), cyclin-dependent kinase (CDK) 6 (ab151247), CDK4 (ab137675), vascular endothelial growth factor (VEGF; ab150766), matrix metalloproteinase (MMP) 2 (ab97779), MMP-9 (ab137867), PI3K (ab180967), phospho (p)-PI3K (ab182651), β-actin (ab8227, all Abcam), AKT (#9272), p-AKT (#9271), JNK (#9252), and p-JNK (#9251, all Cell Signaling Technology, USA). After rinsing, PVDF membranes were incubated with HRP-conjugated secondary antibody (goat anti-rabbit, ab205718, Abcam) at room temperature. An enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA) was used to detect the target proteins. Protein levels were quantified by ImageJ software (National Institutes of Health, USA).

Western blots were performed as previously reported [27 (link), 28 (link)]. Antibodies for p38 (sc-535), JNK1/2 (sc-7345), p-JNK1/2 (sc-6254), ERK5 (sc-398015), p-ERK5 (sc-135760), GAPDH (sc-25778), and goat anti-rabbit IgG-HRP secondary antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for p-p38 (#9211), ERK1/2 (#9102), p-ERK1/2 (#9101), AKT (#9272), and p-AKT (#9271) were purchased form Cell Signaling Technology (Danvers, MA, USA), and goat anti-mouse IgG-HRP secondary antibody was purchased from Bio-Rad (Hercules, CA, USA). The experiments were independently repeated three times.

GEE was provided by NEWTREE Inc. (Kyeonggi, Korea). Its composition is shown in Table 1. Antibodies targeting GLUT4 (sc-7938), phospho-IRS-1 (sc-17196), IRS-1 (sc-559), phospho-PI3K p85α (sc-12929), phospho-c-Jun N-terminal kinases (p-JNK, sc-6254), JNK (sc-571), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-25778) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies targeting PI3K (#4255), phosphoprotein kinase B (p-Akt, #9271), Akt (#9272), phospho-p38 MAPK (p-p38 MAPK, cs-9215), p38 MAPK (#9211), phospho-p44/42 MAPK (p-ERK1/2, #4377), and ERK1/2 (#9102) were purchased from Cell Signaling Technology (Bedford, MA, USA). Metformin was obtained from the Cayman Chemical Company (Ann Arbor, MI, USA).