Fig. 3. Western blotting with anti-MACF1b antibody. (A) Analysis of rabbit polyclonal CU149 antibody. Cell lysate (80 µg) collected from pFLAG-MACF1b-PRD-transfected COS7 cells was subjected to western blotting with mouse monoclonal anti-FLAG antibody (M2), CU149 (149), and preimmune serum of the rabbit that generated CU149 (pres). CU149 and M2 both recognized FLAG-MACF1b-PRD protein (FMP) that was not detected by the preimmune serum. A molecular size standard (kDa) is indicated on the left. (B) Detection of MACF1b in HaCaT cells. HaCaT cell lysate was subjected to immunoblotting with CU149 (MACF1b). Only one slow mobility band was detected. (C) Specificity of CU149 antibody to MACF1b. H460 cells were transfected with MACF1 siRNA vector (MACF siRNA) that was designed to knockdown MACF1a/b, or control vector (Control) for 72 hours. Cell lysates were probed for either MACF1b (top panel) or ß-tubulin (loading control; lower panel). MACF1b protein in cells transfected with MACF siRNA vector was reduced significantly compared to cells transfected with the control vector. (D) Detection of MACF1b proteins in the enriched Golgi fraction. H460 cell lysate was subjected to sucrose step gradient centrifugation, Golgi and ER fractions were isolated, resolved in SDS-PAGE, transferred to PVDF membranes, and probed with CU149 antibody (MACF1b; top panel), anti-p115 antibody (p115; middle panel), and anti-FTCD antibody (FTCD; lower panel). Crude: crude lysate. CU149 only recognizes MACF1b proteins in enriched Golgi fraction of H460 cells. A molecular size standard (233 kDa) is indicated.