Fig. 2. RGS10A silencing blocks RGS10A expression. RAW264.7 cells stably transfected with pAVU-scrambled, pAVU-R10a or pAVU-R10b constructs, and BMMs with pLenti-scrambled or pLenti-R10a were stimulated with RANKL and M-CSF for 96 hours. (A) RT-PCR analysis. Lane 1, negative control (H₂O). The expression of RGS10A mRNA was weak or absent in RGS10A-silenced cells (lane 2, pAVU-R10a; lane 4, pAVU-R10b). (B) Western blotting of RGS10A protein and RGS12 protein in control and RGS10A-silenced RAW264.7 cells (lanes 1-4) and BMMs (lanes 5-7) stimulated with RANKL and M-CSF. For RGS10A protein, the signals were strong in control cells (lanes 1, 2, 5) and weak in RGS10A-silenced cells (lanes 3, 4, 6, 7). However, for RGS12 protein, there is no apparent difference in signals between control cells and RGS10A silenced cells. Lane 1, mock; lane 2, pAVU-scrambled shRNA; lane 3, pAVU-R10a; lane 4, pAVU-R10b; lane 5, pLenti-scrambled shRNA; lane 6, pLenti-R10a; lane 7, pLenti-R10b. Ab, antibody. (C,D) Quantification of RGS10A levels by western blotting as in B. RGS10A protein levels in transfected cells were normalized to the loading control -actin. (E) Immunofluorescence staining. RGS10A expression was silenced in the cells transfected with pAVU-R10a. These are images of phase-contrast as an internal control.