SRB KO destabilizes SRA and disrupts its localization. (A) Schematic representation of CRISPR/Cas9-directed mutations in SRB in HeLa Cas9 (KO1 and KO2) and 293T Cas9 (KO3) SRB KO clonal cell lines. See also Supplemental Figure S1. (B) Representative immunoblot analysis of expression levels and subcellular distributions ([C] cytosol, [ER] membrane fraction, inclusive of endoplasmic reticulum) of SR subunits (SRB and SRA), SRP subunits (SRP54 and SRP72), cytosolic proteins (GAPDH and β-tubulin) and ER-resident proteins (GRP94 and TRAPα) in HeLa and 293T SRB KO clones with paired parental cell line controls. (C) RT-qPCR analysis of SRA and SRB mRNA levels in three SRB KO clones and paired parental cells (WT). Expression levels were normalized to GAPDH with parental cell expression set to 1, n = 3 biological replicates. Data are means ± SD. (*) Indicates P-value ≤0.01. (D) Representative sucrose density gradient polysome profiles of HeLa Cas9 SRB KO2 and paired parental cell lines. See also Supplemental Figure S3. RNA was extracted from gradient fractions of the HeLa Cas9 WT and SRB KO2 cell lines, and semiquantitative RT-PCR was performed for SRB, SRA, and GAPDH mRNAs. PCR products were resolved by agarose gel electrophoresis. Data are representative of three biological replicates.
