Gapdh ab

Cells detached using 200 mg/L EDTA, 500 mg/L trypsin (Cambrex), were lysed in RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 0.1%SDS, 1% Triton X-100, 0.5% NP40) containing protease inhibitor mixture. Protein content of cell lysates was measured by a colorimetric assay (BioRad). 40 μg proteins or 50 μl concentrated conditioned medium from A375 or A375M6 cells were separated on 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. In all cases, the membranes were blocked with 5% non-fat dry milk and probed with 1 μg/mL R4 anti-uPAR monoclonal antibody recognizing uPAR D3 domain, 1 μg/mL anti-FPR1 polyclonal antibody (#128296 Ab, Abcam), 0.2 μg/mL GAPDH Ab (Santa Cruz Biotechnology), or 1 μg/mL 389 anti-uPA polyclonal antibody (American Diagnostica). Washed filters were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and detected by ECL (Amersham- GE Healthcare). Densitometry was performed using the NIH Image 1.62 software (Bethesda,MD). Each experiment was performed three times.

Cells detached using 200 mg/L EDTA, 500 mg/L trypsin (Cambrex), were lysed in RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 0.1%SDS, 1% Triton X-100, 0.5% NP40) containing protease inhibitor mixture. Protein content of cell lysates was measured by a colorimetric assay (BioRad). 30 and 60 μg proteins were separated on 10% SDS-PAGE under unreducing (to detect uPAR) or reducing conditions (to detect FPR1) and transferred onto a polyvinylidene fluoride membrane. Membranes were blocked with 5% non-fat dry milk and probed with 1 μg/mL R4 anti-uPAR mAb, recognizing the DII-DIII uPAR domains, 1 μg/mL anti-FPR1 polyclonal antibody (Ab) (#128296 Ab, Abcam), or 0.2 μg/mL GAPDH Ab (Santa Cruz Biotechnology). Washed filters were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and detected by ECL (Amersham- GE Healthcare). Densitometry was performed using the NIH Image 1.62 software (Bethesda, MD.

The cells were treated with P. dactylifera L. seed extract (0.0245, 0.0368, 0.049, and 0.147 mg/mL) or arbutin (0.54 mg/mL) and then lysed in PBS containing nonidet P—40 (1%), sodium deoxycholate (0.5%), sodium dodecyl sulfate (SDS, 0.1%), aprotinin (5 μg/mL), phenylmethylsulfonyl fluoride (100 μg/mL), pepstatin A (1 μg/mL), and EDTA (1 mM) at 4 °C for 20 min. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (30 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 5% fat-free milk in PBST (PBS with 0.05 % Tween-20) and incubated at 4 °C overnight with the following primary antibodies diluted in PBST: MITF Ab (1:1000), TRP1 Ab (1:6000), TRP2 Ab (1:1000), MC1R Ab (1:500), GAPDH Ab (1:1500), tyrosinase Ab (1:2000), p-p38 Ab (1:500), p38 Ab (1:500), p-JNK Ab (1:500), JNK Ab (1:500), p-ERK Ab (1:500), ERK Ab (1:500), p-CERB Ab (1:500), and CERB Ab (1:200), all from Santa Cruz Biotech (Dallas, TX, USA)). The bound antibodies were detected by horseradish peroxidase-conjugated secondary antibody (Amersham Corp.) followed by ECL detection system (Amersham) according to the manufacturer’s instruction.