Sc 208

Antibodies against the V5 domain of PKCα (sc-208), PKCβI (sc-8049 and sc-209), PKCβII (sc-210) and PKCγ (SC-211) were purchased from Santa Cruz Biotechnology^®. Anti-α-tubulin antibody (T9026) was purchased from Sigma-Aldrich^®. Recombinant proteins PKCα, PKCβI, PKCβII, PKCγ, PKCδ and PKCε produced in Sf9 cells were obtained from Invitrogen^®. Phosphatidylserine (PS) and 1-2-dioleoyl-sn-glycerol (DG) were purchased from Avanti Polar Lipids^® Phorbol myristate acetate (PMA) was obtained from Sigma-Aldrich^®. TMB substrate (3,3′, 5,5′ tetramethylbenzidine) was purchased from BD biosciences^® and secondary antibodies conjugated with Alexa 555 and Alexa 568 were purchased from Molecular Probes^®.

After treatment with desired concentrations of Bis-I, Gö6976, and Rottlerin, cells were harvested and washed in PBS. The extracts were prepared by resuspending cell pellets in lysis buffer (50mM Tris-HCI, pH 8.0, 150 mM NaCI, 1% Triton X-100, 5 mM EDTA), briefly sonicated and centrifuged at 15000xg for 15 min at 4°C. The protein concentrations were determined using BCA assay (Thermo). SDS-PAGE and immunoblotting were performed. Nitrocellulose membranes were probed with anti-PKCα, anti-PKCδ, anti-PKCη, anti-PKCγ, anti-PKCζ, anti-GAPDH (sc-208, sc-213, sc-215, sc-211, sc366126, sc-25778, Santa Cruz Biotechnology), anti-PKCβ, anti-PKCϵ, anti-claudin-1, −3, −4, and −7, anti-CD9, anti-CD82, Tspan-8 (Abcam), anti-caspase 3 and 9, anti-LC3A/B, Anti-Atg-5, anti-EpCAM, anti-E-cadherin, anti-vimentin (Cell Signaling). Immunoblotting signals were developed using chemiluminescence. All experiments were performed in triplicate.

Immunostainings for glial fibrillary acidic protein (ZRF1 from DSHB, 1:200), choline O-acetyltransferase (AB144P from Millipore, 1:500) and protein kinase C alpha (SC-208 from Santa-Cruz, 1:500) were performed after completion of the FISH protocol according to de Oliveira-Carlos et al., 2013 (link). For chat, antigens were retrieved in preheated 10 mM sodium citrate buffer for 6’ at 85 °C. Sections were washed in PBS and 0.3% PBSTx prior to primary antibody incubation. Following the protocol of de Oliveira-Carlos et al., 2013 (link), we washed the sections three times in PBSTx, and incubated in anti-goat or anti-rabbit IgG (H+L) Alexa 488-conjugated secondary antibodies (Invitrogen, 1:750).

To examine the BMP4 protein expression in the retinal tissue, immunohistochemical staining was performed on the human eyes of the donor who died of meningioma. The donor eyes were obtained from the Eye Bank of Guangdong Province. All the procedure was conducted following the Declaration of Helsinki and the written informed content was obtained from the donor before the study. The human eyes were fixed in the 4% paraformaldehyde and then embedded in paraffin. The paraffin-embedded eyes were cut into 4-μm-thick sections. Antigen retrieval was performed on the sections using high temperature (98°C) for 30 min and the sections were blocked with 5% normal goat serum. The primary antibodies, a mouse anti-BMP4 antibody (1:25; sc-12721; Santa Cruz) and a rabbit anti-PKC α antibody (1:500; sc-208; Santa Cruz), were used to incubate the sections. The secondary antibodies were Alexa Fluor 568-conjugated donkey anti-mouse IgG antibody (1:500; ab175472; Abcam) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibodies (1:500; ab150073; Abcam) and DAPI (1:3000; 28718-90-3; Sigma-Aldrich) was used for the nuclear labeling. The images of stained sections were taken using the confocal microscope (Zeiss LSM 980, Carl Zeiss Microscopy GmbH, Jena, Germany).