UPR-reprogramming by aPC depends on p85-sXBP1 signaling. (A) Schematic illustration of homeostatic insulin-dependent ER signaling. (B-C) Representative immunoblots (B, top) and bar graphs (C) showing nuclear levels of sXBP1, ATF6, Chop, and Lamin A/C (loading control) and total sXBP1 levels or phosphorylated IRE1α in whole cell lysates (B, bottom) without or post aPC (20 nM) treatment at indicated time points. (D-E) Treatment with aPC dissociates p85α and p85β protein complexes (D, red, top), while promoting interaction of p85α or p85β with sXBP1 (D, middle and bottom, respectively). Representative immunofluorescence images showing protein complexes (red, PLA). Corresponding line graphs (E) summarizing PLA-positive signals. AU, arbitrary units. PLA complexes (red); nuclear stain DAPI (blue); actin cytoskeleton-phalloidin staining (green). (F) Representative immunoblots showing immunoprecipitates of XBP1 bound to p85α and p85β in resting mouse podocytes without or post aPC (20 nM) treatment at indicated time points. (G) Representative immunoblots showing nuclear levels of sXBP1 in control (control, short hairpin RNA) and p85α and p85β knockdown mouse podocytes without or post aPC (20 nM) treatment at indicated time points. (H-J) DKD in APC^high mice without (Pod^Cre) or with podocyte-specific p85α deficiency or sXBP1 deficiency or constitutive p85β deficiency. Dot plot summarizing albuminuria (H) and representative immunoblots (I) and bar graph (J) showing nuclear levels of ER transcription factors in renal cortex samples. Mean ± SEM of at least 3 independent repeat experiments (C,E) or at least 10 (H) or 8 (J) mice per group. *P < .05; **P < .01 (C,E,H,J: ANOVA). WCL, whole-cell lysates.