Anti pkcα

Immunohistochemistry was conducted as previously described (Coomer and Morris, 2018 (link)) and imaged on a confocal microscope (Leica SP8, Leica). The following antibodies were used: anti-zCDHR1a (CDHR1a, rabbit, 1:100, Bosterbio, Pleasonton, CA, United States), anti-zSiah1 (Siah1, rabbit, 1:100, Bosterbio, Pleasonton, CA, United States), anti-Huc/D (ganglion and amacrine cells, mouse, 1:40), anti-PKCα (bipolar cells, mouse, 1:100, Santa Cruz Biotechnology, Dallas, TX, United States), anti-Prox1 (horizontal cells, rabbit, 1:1000, Acris, San Diego, CA, United States), anti-PCNA (cells in S-phase, mouse, 1:100, Santa Cruz Biotechnology, Dallas, TX, United States), and activated caspase 3 (apoptotic cells). Alexa fluor conjugated secondary antibodies (Invitrogen, Grand Island, NY, United States) and Cy-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:200 dilution and DAPI to label nuclei (1:10,000, Sigma, St. Louis, MO, United States). TUNEL assay was conducted with ApopTag Fluorescin Direct In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, United States) on retinal cryosections according to manufacturer’s instructions.

iMoDCs, plated at 1x10⁵/well in 6-well plates in a final volume of 1 ml, were serum starved for 4 hours and then stimulated in IMDM containing 1% FCS (BioWhittaker) and 20 μg/ml Bet v 1, Api g 1, or a control stimulus for 5, 10, 20, and 30 minutes or left untreated. The control stimulus solution contained 50 ng/ml TNF-α (Strathmann, Hamburg, Germany), 10 ng/ml IL-1β, and 500 U/ml GM-CSF (both PeproTech, Rocky Hill, NJ, USA) and is referred to in the text as maturation-inducing factors (MFs). Cells were lysed in 90 μl Laemmli buffer and the lysates heated for 5 min at 95°C. Protein extracts were separated on 10% SDS gels and transferred to a nitrocellulose membrane (GE Healthcare, Maidstone, UK). Blocking was carried out for 1 hour at room temperature with PBS containing 0.1% Tween 20 (PBST) and 5% skim milk. Primary antibodies (anti-PKCα, anti-pPKCα, anti-Erk1/2 from Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-pErk1/2, anti-pp38, and anti-IκBα from Cell Signaling Technology, Beverly, MA, USA) were diluted in PBST containing 5% BSA and incubated overnight at 4°C. Bound antibodies were visualized by anti-IgG antibodies conjugated with peroxidase (Sigma-Aldrich) and subsequent chemiluminescence detection (LumiGLO, Cell Signaling Technology).

Anti-ASCL1 (MASH1) (1:100, mouse monoclonal, BD Pharmingen, Franklin Lakes, NJ); anti-CHX10 (VSX2) (1:200 goat polyclonal, Santa Cruz Biotechnology, Santa Cruz CA or 1:250 sheep polyclonal, Exalpha Biologicals, Shirley MA); anti-KI67 (1:500 mouse monoclonal, BD Pharmingen, Franklin Lakes, NJ); anti-NESTIN (1:200 rabbit polyclonal, Millipore Temecula CA); anti-PKCα (1:100 rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz CA); anti-RECOVERIN (1:2000 rabbit polyclonal, Millipore, Temecula CA): anti-SOX2 (1:2000 goat polyclonal or 1:100 mouse monoclonal, R and D Systems, Minneapolis MN); anti-ßIII TUBULIN (1:5000 mouse monoclonal, Sigma-Aldrich, St. Louis MO or 1:5000 rabbit polyclonal, Covance, Princeton NJ). All antibodies were validated in previous studies [21 (link),23 (link),30 (link)].

Cells or tissues were collected and lysed in TNES buffer (10 mm Tris, pH 8.0; 150 mm NaCl; 2 mm EDTA; 0.5% SDS). Equal 25 μg aliquots of proteins were electrophoresed on 8% SDS-polyacrylamide gels and electrotransferred onto polyvinylidine difluoride membranes. The blots were blocked with Tris-buffered saline containing 5% non-fat milk for 1 h. The membranes were incubated with specific antibodies: 1 : 1000 anti-TGF-β1, anti-Smad7, anti-SPRY4, anti-PKCα, anti-ERK, anti-JNK, and anti-p38MAPK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and 1 : 1000 anti-p-ERK, anti-p-JNK, and anti-P-p38MAPK (New England BioLabs, Beverly, MA, USA) for 3 h at room temperature, all of which recognize the activated forms of these kinases. Proteins were visualized with peroxidase-conjugated secondary antibodies at 1 : 2000 for 1 h, using an enhanced chemiluminescence detection system (Santa Cruz). Total RNA was collected from RMS cell lines and frozen tissues according to the manufacturer's recommendations. Mature miRNA analysis was performed using TaqMan miRNA assays (Takara Shuzo, Kyoto, Japan), including RT and real-time PCR. RT reactions were performed using a single miRNA-specific stem-loop RT primer as described by Chen et al.^{44 (link), 45 (link)} (Supplementary Table1).

Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-p-AMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti-4EBP1, anti-p-eIF4E, anti-eIF4E, anti-p-P70S6K1, anti-P70S6K1, anti-p-RPS6, anti-RPS6, anti-rictor, anti-p-Akt, anti-Akt, and anti-E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PKC-α, anti-p-Rac1, anti-Rac1, anti-PCNA, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology. (Santa Cruz, CA, USA). Anti-p-rictor was purchased from Millipore (Temecula, CA, USA). Anti-p-PKC-α, anti-F-actin, and anti-Ki-67 were purchased from Abcam (Cambridge, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA) and rapamycin was purchased from Tocris Bioscience (Bristol, UK).

Clematichinenoside (95.3% purity) was provided by China Pharmaceutical University. PD98095, GF109203X, BI-D1870, and KG-501 were purchased from Shang Hai Haoran Biological Technology CO., LTD (Shanghai, China) Dulbecco's modified Eagle's medium (DMEM, high glucose) and newborn calf serum were products of Gibco. Anti-ERK1/2, Anti-phospho-ERK1/2, anti-PKCα, anti-PKCβI/βII, anti-PKCγ, anti-phospho-PKCα, anti-phospho-PKCβI/βII, anti-phospho-PKCγ, anti-p90RSK, anti-phospho-p90RSK, anti-CREB, anti-phospho-CREB, anti-bcl-2, anti-bcl-xl, and anti-bax were purchased from Santa Cruz Biotechnology, Inc. Primers for PCR were obtained from Shanghai Sangon Biotech Co., Ltd. RT-PCR kit was bought from Dalian Takara Biotechnology Co., Ltd.