Fig. 8

Knockdown of SLC35B1 leads to inhibition of BiP activity and to AMPK phosphorylation. a–d HeLa cells were transfected with control siRNA or with SLC35B1-targeting or SLC35B1-UTR-targeting siRNA for 96 h. They were converted to semi-permeabilized cells and tested for protein transport activity as described previously^6,26,38,39. Here, ER protein import, assayed as N-glycosylation, of the small presecretory protein preproapelin²⁶ (which depends on BiP, (a, b), and of the tail anchored Sec61ß²⁶ (which does not depend on BiP, (c, d) were analyzed. The effects of siRNA-mediated depletion of BiP are shown for comparison²⁶. Representative phosphorimages after SDS-PAGE and quantitative data from three independent experiments are reported together with the individual data points as % of control with SEM. Pre, precursor form; g, glycosylated form. e–l HeLa cells were transfected with control siRNA or with SLC35B1- or SLC35B1-UTR-targeting siRNA for 96 h. Where indicated, 0.0001% DMSO, or 10 µM emetine (EM) were present during the last 2 h of growth plus Fura loading. Cells were loaded with Fura-2 for 30 min, transferred to Ca²⁺-free buffer, and Fura-2 signals were recorded as F₃₄₀/F₃₈₀ ratios in real-time^6,27. Where indicated, 1 µM Thapsigargin (Tg), or 5 µM Ionomycin (Iono) were added. e, g, i, k The mean values of the ratiometric recordings are shown with the standard error of the mean (SEM). f, h, j, l Statistical analysis of Tg-induced (f–h) or Iono-induced (j–l) changes in cytosolic Ca²⁺ levels in the experiments with the indicated number of cells in at least three independent experiments. The indicated pairs were assessed by unpaired, two-sided Student´s t-test (*P < 0.05, **P < 0.01, **P < 0.001, ns, not significant). m, n Where indicated, 0.5 mM AICAR was present for 15 h. Cells were treated as indicated and analyzed by SDS-PAGE and Western blotting using phosphorylated AMPK-specific antibodies. Notably, AICAR is an AMP-mimetic and AMPK activator⁴⁶. Staining the blot for ß-actin served as a loading control. Representative luminescence images of the blots and quantitative data from five independent experiments are reported together with the individual data points as % of control with SEM