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. 2010 Nov 16;286(3):1976–1986. doi: 10.1074/jbc.M110.148486

FIGURE 1.

FIGURE 1.

RioK1 is a novel stoichiometric component of the human PRMT5 complex. A, the PRMT5 complex was immunoprecipitated from HeLa extract using anti-PRMT5 antibodies covalently coupled to protein A-Sepharose, resolved via SDS-PAGE, and visualized by silver staining. Normal rabbit serum (NRS) coupled to protein A-Sepharose was used as control (lane 2). The asterisk-marked band at ∼90 kDa was excised and identified as RioK1 by mass spectrometry (lane 3). HC and LC represent the heavy and light chain of co-eluted antibodies. Lane 1 shows protein standard (M). B, the PRMT5 complex was immunoprecipitated from HeLa extract using antibodies directed against PRMT5 (lane 2), WD45/MEP50 (lane 3), pICln (lane 4), RioK1 (lane 5), or normal rabbit serum (NRS) as control (lane 6). The precipitated complex components were analyzed by SDS-PAGE and silver staining. Note that protein content was normalized to PRMT5, explaining the different intensities of co-precipitated pICln in A (lane 3) and B (lane 2). C, the PRMT5 complex was immunoprecipitated similarly to B, resolved by SDS-PAGE, and analyzed by Western blotting using the indicated antibodies. D, HeLa total cell lysate (TCL) was separated by gel filtration chromatography, and fractions were analyzed by Western blotting. E, total protein extracts were generated from MCF7 (breast cancer cell line), HeLa (cervix carcinoma cell line), Kelly (neuroblastoma cell line), NEC8 (testicular tumor cell line), HEK293T (embryonic kidney cell line), MelHo (melanoma cell line), and HS68 (primary foreskin fibroblast cell line) and normalized to β-actin (lowest panel). Expression of RioK1 and the PRMT5 complex components PRMT5, WD45/MEP50, and pICln were analyzed by immunoblotting with specific antibodies as indicated. F, to analyze whether RioK1 is part of the PRMT5 complex in different cell lines, RioK1 was immunoprecipitated from MCF7, HEK293T, and HS68 extracts. Co-precipitation of PRMT5 complex components was assessed by Western blotting with specific antibodies as indicated (lanes 3–5). Normal rabbit serum was used as control (lanes 6–8).