Fig. 7

MANF interacts with PIP4k2b. a Recombinant MANF-His protein was mixed with wild type (WT) mouse brain lysate (MANF/lysate). Two control reactions were set: 1. MANF-His protein only (MANF only); 2. WT mouse brain lysate only (Lysate only). The reactions were incubated at 4 °C overnight, and then Ni-NTA beads were added to pull down MANF-His protein together with its interacting partners. b A single image of silver stain result of His-pull down in Fig. 7a. The band that is specifically present in MANF/lysate is indicated by a black arrow. c Representative western blotting images (from three independent experiments) showed that GST-PIP4k2b was able to precipitate MANF-His. d Co-immunoprecipitation study using PC12 cells transfected with MANF-HA and Myc-PIP4k2b. Representative western blotting images (from three independent experiments) showed that Myc-PIP4k2b was detected in the pull-down lysate. e Co-immunoprecipitation study using MANF transgenic mouse brain lysate. Representative western blotting images (from three independent experiments) showed that PIP4k2b was detected in the pull-down lysate. f Three different GST tagged PIP4k2b fragments (Δ1, Δ2 and Δ3) were generated as demonstrated in the figure. Representative western blotting images (from three independent experiments) showed that fragment Δ1 was able to precipitate MANF-His. g Representative western blotting images (from three independent experiments) of subcellular fractionation using wild type (WT) and MANF transgenic (TG) mouse brain lysate. Fractions of nucleus (nuc), cytosol (cyto), endoplasmic reticulum (ER) and mitochondria (mito) were indicated by antibodies against LSD1, MEK, PDI and COX IV respectively. h Representative western blotting images (from three independent experiments) of PIP4k2b levels in either total or ER fraction of WT and TG mice. i Quantification of western blotting results in Fig. 7h (*P < 0.05, n = 3; 2-tailed student t test, Brain, t = 2.99, P = 0.0480; Liver, t = 1.376, P = 0.3026). Data are represented as mean ± SEM