Fig. 2.

Validation on endogenous MeCP2 reveals known and unknown regulators. The effects of candidate knockdown on endogenous MeCP2 levels were tested in HEK293T cells along with RNA analysis to determine which shRNAs were on-target. For each candidate, three shRNAs (a–c) were transfected along with vectors expressing scrambled shRNA and shRNA targeting MECP2. Two days post-transfection, puromycin was applied to select for cells expressing the silencing plasmid. Selection was maintained for four days then cells were split for parallel immunoblot analysis (top) and RNA analysis (bottom). (A) The effects of Protein Phosphatase 2 Regulatory subunit 1 Alpha (PPP2R1A) knockdown on endogenous MeCP2 levels relative to GAPDH. Coupled RNA analysis (below) indicated that only the shRNAs “a” and “c” which resulted in significant knockdown of PPP2R1A significantly decreased MeCP2 protein levels. (B) As in A, but for Homeodomain Interacting Protein Kinase 2 (HIPK2). (C) As in A and B, but for Homeodomain Interacting Protein Kinase 1 (HIPK1). (D) As in A–C, but for RIO Kinase 1 (RIOK1). K.D., knockdown. n = 3–4 per group. *p< 0.05; **p<0.01; ***p<0.001, two-tailed t-test.