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. 2013 Oct 17;288(48):34599–34611. doi: 10.1074/jbc.M113.503128

FIGURE 3.

FIGURE 3.

Expression or co-expression of ASL wild type and transcript variants in different mammalian cell lines and human livers. Western blot analysis of ASL expression in cell extracts of COS-1, 293T, and HeLa cells (A); in human livers (B); or in transfected 293T cells (C–G). 30 μg of total protein was separated by 10% SDS-PAGE (A, B, D, F, G, and top panel in C) or by native PAGE (E and middle panel in C) for analyzing ASL expression. A, NT, non-transfected; wt, pcDNA3-ASL-WT (P-WT). B, lanes 1–3, human liver samples from three controls. C, ASL expression in 293T cells non-transfected (NT) or transfected with EV, P-WT, pcDNA3-ASL-ex2del (P-ex2del), or pcDNA3-ASL-ex7del (P-ex7del), respectively. D, (co-)expression of ASL monomers in 10% SDS-PAGE (ASL-WT, 52 kDa; ASL-ex2del, 45 kDa). Lane 1, P-WT with EV; lane 2, P-WT with P-ex2del and EV; lane 3, P-WT with P-ex7del and EV; lane 4, P-WT with P-ex2del and P-ex7del; lane 5, P-ex2del; lane 6, P-ex7del; lane 7, EV. E, (co-)expression of ASL homotetramers in 10% native PAGE (ASL-WT, ∼200 kDa; ASL-ex2del, ∼180 kDa). Lanes 1–7, same as in D. Asterisks, nonspecific bands. F, ASL expression in 293T cells co-transfected with P-WT and P-ex2del at different ratios. Lane 1, P-WT (1.75 μg); lane 2, 1:1; lane 3, 1:2; lane 4, 1:5; lane 5, P-WT (3.5 μg); lane 6, 2:1; lane 7, P-WT (8.75 μg); lane 8, 5:1; lane 9, ASl-ex2del (3.5 μg). G, (co-)expression of ASL mutant p.E189G (P-E189G) with transcript variants. Lane 1, P-WT with EV; lane 2, P-E189G with EV; lane 3, P-WT with P-E189G; lane 4, P-WT with P-ex2del; lane 5, P-WT with P-ex7del; lane 6, P-E189G with P-ex2del; lane 7, P-E189G with P-ex7del; lane 8, P-ex2del; lane 9, P-ex7del. Asterisks, nonspecific bands. β-Actin (42 kDa) and GAPDH (37 kDa) served as loading control.