Figure 4. SRP mediated targeting to the ER ensures efficient spicing of XBP1u mRNA.
(A) HEK 293 cells were transfected with shRNAs against luciferase (control), SRP14, or SRP54. 5 days after transfection, the cells were replated for transfection with XBP1u and treated with 10 mM DTT for the indicated time periods. The Trizol harvested cells were analyzed as in Figure 2D. A phos-tag gel was used for the Ire1α immunoblot. p-Ire1α and p-PERK indicate the phosphorylated forms of Ire1α and PERK, respectively. * denotes a background band. (B) HeLa cells were transfected with control siRNA or siRNA targeting the α subunit of the SRP receptor (SRα) and analyzed as described in Figure 2D. (C) HeLa cells were transfected with the indicated siRNA oligos and treated with 10 mM DTT for 2 hr after 96 hr post-transfection. Cells were harvested in SDS sample buffer and analyzed for IB with indicated antibodies. Upon DTT treatment, the ATF6α band disappears due to the cleavage of its N-terminal cytosolic domain. Our ATF6α antibodies were not suitable for detecting the cleaved N-terminal cytosolic domain (not shown). Note that depletion of Sec61α caused significant reduction of the transmembrane proteins PERK and ATF6α.