Figure 3: Effect of PKA inhibitor H-89 on the expression of LHR mRNA and LRBP protein in human granulosa cells. Day 3 granulosa cells were serum-starved, treated with hCG (10 IU/ml) with or without the PKA inhibitor H-89 (10 μM; 1 h pretreatment) for a total of 12 h, and were either processed for total RNA isolation or lysed using RIPA buffer. A. Total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for LHR. The graph represents changes in mRNA levels normalized to 18S rRNA, shown as fold change vs. control. Error bars, mean ± SE. *, P < 0.05 vs. control CTL; #, P < 0.05 vs. hCG; n = 4. B . Cell lysates were subjected to Western blot analysis to detect LRBP using LRBP antibody. The membranes were then stripped and reprobed for â-tubulin. The lower panel represents densitometric scanning of the LRBP normalized for tubulin and expressed as fold change vs. CTL. The blot shown is representative of three independent experiments, and the results in the bar graph are average and SE of three experiments. *, P < 0.05 vs. CTL; #, P < 0.05 vs. hCG. (Source: Ref 27, Reproduced with permission).