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. 2020 Oct 12;21(20):7523. doi: 10.3390/ijms21207523

Figure 6.

Figure 6

Effect of mitogen-activated protein kinase (MAPK) inhibition on EGR-1 expression. (A) Serum-starved MCF-7 cells (cultured in medium containing 0.5% serum for 24 h) were treated with 10 ng/mL TNFα for various durations (0–60 min). Total protein extracts were analyzed by immunoblotting using antibodies against phospho- extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), phospho- c-Jun N-terminal kinase 1/2 (JNK1/2) (Thr183/Tyr185), and phospho-p38 kinase (Thr180/Tyr182). Corresponding total MAPK protein was used as an internal control. (B–D) Serum-starved MCF-7 cells were pretreated with 5 μM U0126, 10 μM SP600125, or 10 μM SB203580 for 30 min, followed by treatment with 10 ng/mL TNFα. After 60 min, cells were harvested and EGR-1 expression was examined by immunoblotting (B), RT-PCR (C), and qR-PCR (D). GAPDH was used as an internal control. Band intensities were measured using the ImageJ software. Bars represent the mean ± SD (n = 3). *** p < 0.001 by Dunnett’s multiple comparisons test. qR-PCR, quantitative real-time PCR.