P plcγ2 4511

Cell lysates were separated by SDS‐PAGE and electrophoretic transferred onto the PVDF (polyvinylidene difluoride) membranes (BIO‐RAD). Transferred membranes were blocked with 5% skim milk in TBS with 0.1% Tween‐20, and then incubated with primary antibodies at 4°C overnight. Following day, membranes were washed three times in TBS buffer with 0.1% Tween‐20 and incubated with the secondary antibody for 45 min at room temperature. Membranes were washed five times in TBS buffer with 0.1% Tween‐20 each 5 min then imaging by ChemiDoc Imaging system (BIO‐RAD). The following antibodies were purchased from Cell Signaling Technology [(VEGFR2‐ #9698, p‐AKT S473‐ #4060, p‐p44/42‐ #9101, p‐p38‐ #3871, p‐PLCγ2‐#4511, neuropilin 1‐ #3725)]. Antibodies to β_IV‐spectrin and β‐actin were purchased from Santa Cruz Biotechnology (#SC514744) and Sigma‐Aldrich (#A1978) respectively. VEGFR1 antibody was purchased from R&D Systems (#AF471).

Cell lysates were separated by SDS‐PAGE and electrophoretic transferred onto the PVDF (polyvinylidene difluoride) membranes (BIO‐RAD). Transferred membranes were blocked with 5% skim milk in TBS with 0.1% Tween‐20, and then incubated with primary antibodies at 4°C overnight. Following day, membranes were washed three times in TBS buffer with 0.1% Tween‐20 and incubated with the secondary antibody for 45 min at room temperature. Membranes were washed five times in TBS buffer with 0.1% Tween‐20 each 5 min then imaging by ChemiDoc Imaging system (BIO‐RAD). The following antibodies were purchased from Cell Signaling Technology [(VEGFR2‐ #9698, p‐AKT S473‐ #4060, p‐p44/42‐ #9101, p‐p38‐ #3871, p‐PLCγ2‐#4511, neuropilin 1‐ #3725)]. Antibodies to β_IV‐spectrin and β‐actin were purchased from Santa Cruz Biotechnology (#SC514744) and Sigma‐Aldrich (#A1978) respectively. VEGFR1 antibody was purchased from R&D Systems (#AF471).