P tyr

Protein lysates of cells growing as a monolayer were prepared as described previously [40 (link)]. Protein concentrations were determined by the Bradford assay (Biorad). 30 μg of protein were loaded on a 4–12% Bis-Tris gel (Invitrogen) and blotted onto a nitrocellulose membrane.
Immunohistochemistry of paraffin-embedded sections was performed as described previously [45 (link)]. Antigen-retrieval consisted of microwaving in 0.01 M citrate buffer (pH 6.0) for 10 min. Immunoperoxidase-based detection was performed using the Histostain-Plus 3rd Gen IHC Detection Kit (Invitrogen/Thermo Fisher Scientific) according to manufacturer's recommendations. Cells were analyzed using an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera.
Primary antibodies used for immunoblotting and immunohistochemistry were ABL1, CDK2, pTyr (all Santa Cruz), actin (Sigma), pABL1 Y412, pAKT S473, AKT, pCDK2 T160, cleaved caspase 3, pCRKL Y207, CRKL, pKIT Y719, pMAPK p42/44 T202, pPDK1 S241, PDK1, PP2A, pS6K T389, S6K (all Cell Signaling Technologies), CIP2A, PHLPP, SET (all Bethyl Laboratories), cyclin A (Novocastra), KIT (DakoCytomation) and MAPK (Invitrogen/Thermo Fisher Scientific).

All chemical reagents were obtained from Sigma (St. Louis, MO, USA), except where noted. 2′,7′-Dichlorodihydrofluorescein was from Molecular Probes, Inc. (Eugene, OR, USA). Western blotting detection reagents were purchased from Amersham (Bucks, UK). RNAzol^TM B was obtained from TEL-TEST, Inc. (Friendwood, TX, USA). Antibodies against catalase, MnSOD, β-actin, Histone H1, p-ser, p-tyr, p-Akt, total-Akt, NF-kB, p-Pak1, total-Pak1, iNOS, and COX-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against FoxO6, p-FoxO6 (Ser184) were obtained from Dr. H. H. Dong (University of Pittsburgh, PA). Anti-rabbit IgG-horseradish peroxidase-conjugated antibody and anti-mouse IgG-horseradish peroxidase-conjugated antibody were from Amersham (Bucks, UK). Horseradish peroxidase-conjugated donkey anti-sheep/goat IgG was purchased from Serotec (Oxford, UK). Polyvinylidene difluoride (PVDF) membranes were obtained from the Millipore Corporation (Bedford, MA, USA).

Total proteins were extracted with Igepal CA-630 lysis buffer containing 50 mMTris-HCl pH 7.5, 150 mMNaCl, 0.5% Igepal CA-630, 1 mM Na₃VO₄, 25 mMNaF, 2 mM PMSF, 8 nMAprotinin; enriched plasma membrane proteins, used for both mass spectrometry and immunoprecipitations analyses, were obtained using Mem-PER Eukaryotic Membrane Protein Extraction Kit (Pierce). For CD98hc immunoprecipitation, cell extracts were incubated overnight at 4° C with CD98hc antibody and then overnight with 20 μl of protein A/G Plus-Agarose (Santa Cruz Biotechnology). Pellets were washed five times with 1 ml of lysis buffer, resuspended in Laemmli sample buffer 2× and subjected to SDS-poly-acrylamide electrophoresis (SDS-PAGE) [58 (link)]. For PTPRJ immunoprecipitation, MagneHIS Protein Purification System (Promega) was used as described above. For Western blot analysis, proteins (50 μg) were loaded and separated on polyacrylamide gels and transferred to nitrocellulose filter membranes. Membranes were blocked in 5% non-fat dry milk, incubated with primary PTPRJ (R&D Systems), CD98hc (Cell Signaling Technology), p-Tyr, Ubiquitin, γ-tubulin, α-tubulin and GAPDH antibodies (Santa Cruz Biotechnology), detected by the appropriate secondary antibodies (Santa Cruz Biotechnology) and revealed by enhanced chemiluminescence (ECL; Amersham Inc.).

Immunoblotting was performed as previously described [11 (link)]. Briefly, muscles were denatured in lysis buffer and protein concentrations were measured using a BCA assay kit (#A53225, Thermo Fisher Scientific, Waltham, MA, USA). A total of 25 µg of protein lysate was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. Primary antibodies, including mTOR (1:1000, #2983), p-mTOR (Ser2448) (1:1000, #2971), rps6 (1:1000, #9272), p-rps6 (Ser473) (1:1000, #9271), ubiquitin (1:1000, #3933S), ATG7 (1:1000, #8558, Cell signaling technology, MA, USA), LC3B (1:1000, #NB100-2220, Novus Biologicals, CO, USA), PGC1-α (1:1000, ab54481, abcam, Cambridge, UK), MuSK (1:1000, sc-134398), p-Tyr (1:1000, sc-508, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-tubulin (1:5000, #05-661, Merck Millipore, Burlington, MA, USA), were incubated overnight at 4 °C. Then, the membrane was washed with TBS-T and incubated with a secondary antibody at room temperature for 1 h. After immunodetection using ECL detect reagent (GE healthcare, Buckingham, UK), protein was quantified using ImageJ image analysis software (National Institutes of Health).