Phospho erk1 2 thr202 tyr204

TDSCs were lysed in lysis buffer containing protease and phosphatase inhibitors (Keygentec, Nanjing, China). Protein concentration was quantified by BCA kit (Keygentec), and an equal amount of protein was loaded in each lane. Constant voltage electrophoresis was carried out with 10% polyacrylamide gels. Then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck, Massachusetts, USA). PVDF membranes were blocked with 3% BSA (Sigma) and hybridized with ERK1/2 (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology), FAK (Santa Cruz Biotechnology), phospho-FAK (Tyr397) (Santa Cruz Biotechnology), and β-actin (Santa Cruz Biotechnology) primary antibodies overnight at 4°C. After being washed with TBST (Keygentec), the PVDF membranes were incubated with secondary antibodies (ZSGB-bio, Beijing, China) at room temperature for 30 minutes, followed by washing with TBST. Finally, the PVDF membranes were covered with 3,3-diaminobenzidine (DAB) for the display of specific protein bands.

The chemicals used were purchased from the following sources: rabbit polyclonal VEGFA (sc-507) and phospho-ERK1/2 (Thr202/Tyr204; sc-16982-R); mouse monoclonal Flk1 (sc-6251; VEGFR2), ERK1/2 (sc-135900), and α/β tubulin (sc-51502); goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (sc-2004) and goat anti-mouse IgG conjugated to HRP (sc-2005) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal phospho-VEGFR-2 (pY1173; #2478), rabbit polyclonal phospho-PLCγ1 (Tyr783; #2821), and PLCγ1 (#2822) were purchased from Cell Signaling Technology (Beverly, MA, USA). SuperSignal West Pico Chemiluminescent substrate (34080) and RNAlater™ Stabilization Solution (AM7020) were purchased from Thermo Scientific (Rockford, IL, USA); RNeasy Mini Kit was purchased from Qiagen (Chatsworth, CA); ThermoScript™ RT-PCR Transcription kit was purchased from Invitrogen (Milan, Italy); PowerUp SYBR™ Green Master Mix was purchased from Life Technologies (Carlsbad, CA, USA). Primers were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA). Eosin–Floxin alcoholic solution (05-10020/L) and Carazzi's Hematoxylin–Nuclear staining (05-06012/L) were purchased from Bio-Optica (Bio-Optica Milano SpA, Milan, Italy). All other reagents were obtained from Sigma-Aldrich Company (St. Louis, MO, USA).

HEK293 cells transiently co-expressing hIR–Rluc8 and Grb2–Venus were used for ERK1/2 and Akt activation using the classical SDS-PAGE and western blotting technique as well as the homogenous time-resolved fluorescence (HTRF^®)-based assay (CisBio Bioassays, Codolet, France) as previously described (31 (link), 32 (link)), respectively. For SDS-PAGE and western blot, cells were first cultured in 6-well plate and starved overnight in serum-free DMEM. After pre-treatment or not with 1 ml/well of defatted camel milk for 30 min at 37°C, cells were then stimulated or not with 100 nM or 1 μM of insulin in 500 μl/well of PBS for 5 min at 37°C. Protein extraction and western blotting were then performed using the rabbit polyclonal antibody against phospho-ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1/1000. The western blot signals were quantified with GeneTools software (release 4.01.02) and expressed in arbitrary units after normalization. In HTRF^®-based assay, the two-plate protocol was used as recently described (32 (link)) after pre-treatment or not with defatted camel milk and stimulation or not with 100 nM insulin at the indicated times. The plate were then incubated for 2 h at room temperature before reading the fluorescence emission at 620 and 665 nm using the appropriate HTRF programs on Mithras LB 943 plate reader.