Vangl2

For IF, paraffin-embedded sections were stained as deScribed previously^{40 (link)} using the following primary antibodies: TAZ (Cell Signaling, 1:1000), K18 (Sigma, 1:200), αSMA (Sigma, 1:250), Ki67 (Abcam, 1:1000), caspase 3 (Cell Signaling, 1:50), AQP (Calbiochem, 1:100; Darmstadt, Germany), Scrib (Santa Cruz, 1:100), Vangl2 (Santa Cruz, 1:50), GM130 (BD Bioscience, 1:150), Sox9 (Millipore, 1:100), NKCC and Slug (Santa Cruz, 1:100). Alexa 488 and Alex 568-conjugated secondary antibodies (ThermoFisher Scientific, Molecular Probes, Waltham, MA, USA) were used at 1:1000. DAPI (6-diamidino-2-phenylindole) was used to counterstain nuclei. Images were acquired using Zeiss Axiovert 40CFL microscope. For IHC, the following primary antibodies were used: vimentin (BD Bioscience, 1:200), LKB1 (Cell Signaling, 1:100), BMP4 (Abcam, 1:100), sox2 (Abcam, 1:100), and ALDH (BD Bioscience, 1:50). For detection, the ImmPRESS REAGENT KIT (Vector Laboratories, MP-7500) was used followed by the DAB kit (Vector Laboratories, SK-4100) and counterstained with hematoxylin (Vector Laboratories, H-3041).

Hippocampal tissue homogenization, protein separation, and transfer to polyvinylidene difluoride membranes were performed as previously described (Havekes et al., 2012 (link)). Membranes were blocked in 5% BSA or 5% non-fat milk in TBST and incubated with primary antibodies (translin, 1:100,000 for total hippocampal lysates, 1:1000 for synaptosomes; trax, 1:1,000; myc, Cell Signaling, Cat. #: 2276S (RRID:AB_331783), 1:5,000; ALK7, Millipore, Cat. #: 09–158 (RRID:AB_1163378), 1:1,000; VANGL2, Santa Cruz, Cat. #: sc-46561 (RRID:AB_2213082), 1:200) overnight at 4°C. Membranes were washed and incubated with appropriate horseradish peroxidase-conjugated goat anti-mouse, anti-rabbit or donkey anti-goat IgG (Santa Cruz, 1:1,000) for 1 hr in room temperature. Blots were exposed on a film by ECL (Pierce, Cat. #: 32106) and quantified using ImageJ. The density of signal was normalized to β-tubulin levels (Sigma, Cat. #: T8328 (RRID:AB_1844090), 1:50,000) for total hippocampal lysates, or to synaptophysin levels (Millipore, Cat. #: MAB368 (RRID:AB_94947), 1:2,000) for synaptosomes. The mean protein level of each group was normalized to the mean protein level of the control WT group (100%).