P38 ab

Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4 °C for 20 min. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF (poly(vinylidene fluoride)) membrane. The membrane was blocked in 5% fat-free milk in PBST (PBS with 0.05% Tween-20), followed by incubation overnight with the following primary antibodies diluted in PBST: JNK Ab, p-JNK Ab, p38 Ab, p-p38 Ab, ASC Ab, procaspase-1 Ab (diluted to 1:1000, all from Santa Cruz Biotech (Dallas, TX, USA)), p65 Ab (Genetex, Taiwan) and NLRP3 Ab (Adipogen, San Diego, CA, USA). The primary antibodies were removed, and the membrane was washed extensively in PBST. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:20,000, Santa Cruz Biotech) was performed at room temperature for 2 h. The membrane was washed extensively in PBST to remove any excess secondary antibodies, and the blot was visualized with enhanced chemiluminescence reagent (GE Healthcare, South Jakarta, Indonesia).

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.

Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4°C for 20 minutes. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF membrane. The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05% Tween-20) followed by incubation overnight with the following primary antibodies diluted in PBST buffer: TM antibody (Ab), MMP-2 Ab, JNK Ab, pJNK Ab, p38 Ab, pp38 Ab, c-jun Ab, c-fos Ab, lamin B1 Ab (diluted used in 1 : 1,000, all from Santa Cruz Biotech [Dallas, TX]), MMP-9 Ab (Abcam, Cambridge, UK), p65 Ab, and p53 Ab (Genetex, Irvine, CA). The primary antibodies were removed, and the membrane was washed extensively in PBST buffer. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1 : 20,000, Santa Cruz Biotech) was performed at room temperature for 2 hours. The membrane was washed extensively in PBST buffer to remove any excess secondary antibodies, and the blot was visualized with enhanced chemiluminescence reagent (GE Healthcare).