Alexa labeled secondary antibodies

To visualize the subcellular localization of inducible or endogenous PPM1A and YAP, or transfected YAP-HA, LATS1-HA, and PRP4K-HF, HEK293A cells were treated as indicated or transfected with specified plasmids for 24 hours before harvest. In the case of Edu staining of intestinal organoids, 50 μM Edu (RiboBio) was added to the culture media for 2 hours before fixing organoids. The cells/organoids were fixed in 4% paraformaldehyde, blocked in 2% bovine serum albumin in PBS for 1 hour, and incubated sequentially with primary antibodies anti-HA (3724S, Cell Signaling Technology, 1:200 dilution), anti-Flag (F3165-5MG, Sigma-Aldrich, 1:500 dilution), anti-Myc (2276, Cell Signaling Technology, 1:100 dilution), anti-YAP (sc-101199, Santa Cruz Biotechnology, 1:100 dilution) or anti-YAP (14074, Cell Signaling Technology, 1:100 dilution), or anti-PPM1A (3549, Cell Signaling Technology, 1:200 dilution) and Alexa-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania, USA, 111-095-003; 115-095-003; 111-025-003; 115-025-003, 1:500 dilution) with extensive washing. Slides were then mounted with VectaShield and stained with DAPI (Vector Laboratories, Burlingame, California, USA). Immunofluorescence images were obtained and analyzed using the Nikon Eclipse Ti inverted microscope or by the Zeiss LSM710 confocal microscope.

Primary antibodies: anti-activated-caspase 3 (Abcam 2302), anti-beta-actin (Sigma A1978), anti-CD31 (BD Biosciences 553370), anti-Ki67 (Abcam 15580), anti-Met (D1C2; Cell Signaling 8198), anti-phospho-Met (Tyr1234/1235; Cell Signaling 3077), anti-Plexin-B1 (A-8; Santa Cruz sc-28372), anti-Plexin-B2 (R&D AF5329), anti-Plexin-B3 (R&D AF4958), anti-Sema4C (LSBio LS-C168955), anti-Vinculin (Sigma V9131).
For Western blotting, protein lysates were prepared with RIPA buffer (Sigma) containing protease and phosphatase inhibitors. Secondary antibodies IRDye 680 or 800 (LI-COR) were used for blot detection with Odyssey Infrared Imaging System (LI-COR). For immunofluorescence labeling of cells and tissue sections, Alexa-labeled secondary antibodies (Jackson ImmunoResearch) and DAPI (Invitrogen) nuclear staining were used.
Tissue microarray analysis was performed with arrays T175 and GL803a (US Biomax) containing cores from glioblastoma (n=34), astrocytoma (n=21), oligodendroglioma (n=10), oligo-astrocytoma (n=4), and normal brain (n=7). Tissues were subjected to heat-induced epitope retrieval with Basic Antigen Retrieval Reagent (R&D), stained for immunosignals with DAB kit (R&D), and counterstained with Gill's #2 hematoxylin.