Extended Data Fig. 5: HG engages several downstream effectors leading to β-cell proliferation (related to Fig. 8).
From: In vivo screen identifies a SIK inhibitor that induces β cell proliferation through a transient UPR
(a) Time-course of the Tm effect on ATF6-N and BIP protein levels in MIN6 cells. Left panel: Representative immunoblot with GAPDH used as a loading control. Right panel: Quantification of ATF6-N and BIP relative protein expression over time. N = 3 independent experiments per time point. (b) Immunoprecipitation experiments to examine a potential direct interaction of ATF6 with SIK1. Upper panel: Experimental workflow. HEK cells were transfected with a Myc-Flag-SIK1 plasmid and treated with DMSO or HG for 6 h. After incubation of cell lysates with anti-Flag antibodies and immunoprecipitation, samples were immunoblotted using anti-ATF6 or anti-Flag antibodies. Lower-left panel: The negative control corresponds to non-transfected HEK cells (Neg). The input displays an ATF6 band that is not detected in the samples after immunoprecipitation with anti-Flag (SIK1). Lower-right panel: The input and IP show the presence of Flag-tagged SIK1 in the samples. One representative experiment out of 2 is shown. (c) Immunoprecipitation experiments to examine the phosphorylation of ATF6 at Ser/Thr residues. Upper panel: Experimental workflow. HEK cells were transfected with a Flag-ATF6 plasmid and treated with DMSO or HG for 6 h. After incubation of cell lysates with anti-Flag antibodies and immunoprecipitation, samples were immunoblotted using anti-phospho-Ser/Thr or anti-ATF6. Lower-left panel: The negative control corresponds to IP lysis buffer (Neg). The input shows phosphorylation marks (indicated by arrows, ph) in both DMSO or HG-treated cells, marks that are no longer detected in the samples after immunoprecipitation with anti-Flag (ATF6). Lower-right panel: The input and IP show the presence of an ATF6 band in the samples. One representative experiment out of 2 is shown.
