Anti numb

After stimulation with LPS at indicated time points, whole cell lysates were extracted using RIPA lysis buffer containing protease inhibitor cocktail and a phosphatase inhibitor (Roche, Germany). Protein concentrations were measured using a BCA Protein Assay kit (Pierce, USA) following the manufacturer’s instructions. Antibodies used in this study were anti-Notch1 (clone C-20), anti-GAPDH (Santa Cruz Biotechnology, USA), anti-ITCH (Epitomics, USA), anti-cleaved Notch1 (Val1744), anti-Numb, anti-phospho NF-κB p65, anti-NF-κB p65, anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-p42/44 MAPK, anti-p42/44 MAPK, anti-phospho-SAP/JNK MAPK, anti-SAP/JNK MAPK, anti-pAkt (Thr308), anti-Akt, HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, USA).

General laboratory chemicals were obtained from Sigma-Aldrich or Merck Millipore. Anti-β-ACTIN and anti-HA antibodies were purchased from Sigma-Aldrich. Anti-H3K27me3, anti-EZH2, anti-JMJD3, anti-MUSASHI (MSI), anti-NUMB, anti-NOTCH1, anti-Cleaved Notch1 (Val1744) (NICD), anti-Tyr485 p85/ Tyr199 p55 phospho-PI3K, anti-Ser2448 phospho-mTOR, anti-Thr389 phospho-p70S6K and anti-Ser536 phospho-NF-κB p65 were purchased from Cell Signaling Technology. Anti-ADRP, anti-CD36, anti-ABCA1, anti-SMRTe and anti-MINT (SPEN) were purchased from Santa Cruz Biotechnology, Inc. HRP conjugated anti-rabbit IgG and anti-mouse IgG and anti-rabbit DyLight 488 were obtained from Jackson ImmunoResearch. Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) to mouse MHC class II, phycoerythrin (PE)-conjugated mAbs to mouse F4/80 were from BD Biosciences. Anti-mouse CD19-APC and CD3-FITC were from Imgenex. Ziehl-Neelsen (ZN) staining Kit was purchased from HiMedia and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was from Sigma-Aldrich. BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) and HCS LipidTOX Red neutral lipid stain was from Molecular Probes (Invitrogen/Thermo Fisher Scientific).

Cells and kidney tissues were lysed with RIPA buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10mM Tris pH 8.0 and 140mM NaCl, 1% NP-40, 0.1% SDS, 100 mg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail), and lysates were subjected to Western blot analysis using method described previously [44 (link)]. The following primary antibodies were used in this study: anti-α-SMA and anti-fibronectin (Sigma-Aldrich, St. Louis, MO, USA); anti-collagen I (Calbiochem, EMD Biosciences, Darmstadt, Germany); anti-p53, anti-p21 and anti-cyclinB1 (Santa Cruz Biotechnology, CA, USA); anti-JNK, anti-p-JNK, anti-p-p53, anti-TGF-β1, anti-Numb and anti-cyclinD1 (Cell Signaling Technology, Beverly, MA, USA); anti-CTGF (Abcam, Cambridge, UK).

Total extracts and immunoprecipitations were performed as described previously.^{33 (link)} For immunoblotting, proteins were resolved by SDS-PAGE and blotted into the nitrocellulose membrane. The blots were incubated with the following antibodies: anti-β-actin, anti-HA, anti-myc and anti-flag were from Sigma-Aldrich; anti-tubulin, anti-p53, anti-p21, anti-Mdm2 and anti-laminB were from Santa Cruz Biotechnology; and anti-numb, anti-phospho-PKCθ (Thr538) and anti-P-ser PKC substrate were from Cell Signaling.

Fetal thymi were fixed with 4% paraformaldehyde solution in PBS, treated with 20% sucrose solution in PBS, immersed in OCT (Tissue Tek) and frozen with a mixture of 2-methylbutene and dry ice. Sections (10 μm) were fixed, permeabilized with Triton X-100, blocked in a solution containing 5% normal goat serum and 3% of bovine serum albumin and stained in the same solution. Sections were mounted with ProLong Antifade (Molecular Probes, Life Technologies Corporation, Carlsbad, CA, USA). Antibodies used were as follows: anti-numb (Cell Signaling, Beverly, MA, USA), anti-CD44 Alexa-647 conjugated, anti-pTα (BD Bioscience), biotinylated anti-myc (Upstate Biotechnology) and DAPI. We use a Leica TCS SP5 confocal microscope with either x20 or x40 objective; images were collected at 8-bit depth, with a resolution of 1024 × 1024 pixels. Images were processed using LAS AF (Leica Microsystems) and Adobe Photoshop software (Adobe Systems, San Jose, CA, USA). The software LAS AF (Leica Microsystem) was used to quantify fluorescent images. Inside each cell, one gate for the nucleus, using DAPI as a marker, and one gate for the cytosol was drawn and threshold intensities of total pixels were determined for Numb.

To extract the protein from patient samples, normal and tumor tissues were homogenized in RIPA buffer containing 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA-free protease inhibitor cocktail (Roche, Germany) plus 1.5 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were collected following the removal of insoluble material from tissue extracts by centrifugation at 14,000 rpm for 20 min at 4°C. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel transfer to a nitrocellulose membrane (BioRad, USA). The membranes were incubated first with the primary antibodies, and then with secondary antibodies coupled to horseradish peroxidase (HRP). Band signals were detected with an enhanced chemiluminescence (ECL) system (Thermo Scientific) and visualized by image analyzer (Fujifilm, Japan). The primary antibodies used for this study are anti-GAPDH (Kang Cheng Bio-tech, China), anti-QKI (Sigma, USA), anti-γ-tubulin (Sigma, USA), anti-FLAG (Sigma, USA), anti-NUMB (Cell Signaling, USA), anti-NICD (Val1744, Cell Signaling, USA), and anti-HA (Roche, Germany). The HRP-conjugated secondary antibodies anti-mouse IgG and anti-rabbit IgG were purchased from Promega.