Anti rb

Anti-PARP (#9542), anti-Bcl-xL (#2762), anti-Bcl-2(#2876), anti-GAPDH (#2118), anti-cyclin D1 (#2922), anti-phospho-Rb(S807/811) (#9308), anti-Rb (#9309), anti-cleaved caspase 3 (#9661), anti-phospho-p70 S6 kinase(Thr389) (#9205), anti-S6 (#2217) and anti-phospho-S6(Ser240/244) (#5364) antibodies were from Cell Signaling Technology. Anti-phospho-STAT3(Y705) (sc-8059), anti-STAT3 (sc-7179), anti-phospho-Erk(T204) and anti-Mcl-1(sc-819) antibodies were from Santa Cruz Biotechnology. Anti-Erk (#06-182) and anti-gp130 (#06-291) antibodies were from Upstate. Anti-α-tubulin (T5168) antibody, water-soluble cholesterol (C4591) and Tyron were from Sigma Aldrich. Anti-Ki67 (#RM-9106-R7) antibody was from Thermo Scientific. Horseradish peroxidase-labeled secondary antibodies were from Jackson ImmunoResearch Laboratories. Secondary HRP-Polymer MACH3 (#M3RS31) antibody was from BioCare Medical. Secondary ImmPRESS (#MP-7401) antibody was from Vector Laboratories. Amplex Red Cholesterol assay kit (A12216) was from Molecular Probes. Recombinant IL-6 was from Pepro Tech. EUK207 was from Shirichai Orian, UCLA. MEDICA analog [HOOC-C(CH₃)₂-(CH₂)₁₂-C(CH₃)₂-COOH] was synthesized as previously described^{11 (link)}. Bortezomib was from Janssen Pharmaceutica.

Paraffin-embedded xenografts or patients’ tissue samples were incubated for 2 h at 56°C for deparaffinization. Antigens were retrieved by microwave treatment in citrate buffer for 15 min to restore antigenicity. After peroxidase activity was blocked with 3% H2O2/methanol for 10 min, sections were incubated with normal goat serum for 20 min to block non-specific antibody binding sites. Sections were incubated with the primary antibodies for 1 h at 25°C and then with biotinylated anti-rabbit/mouse IgG and peroxidase-labeled streptavidin for 10 min each. The following primary antibodies were used: Rabbit anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4 and anti-CCND1 (1:100, Cell Signaling Technology), anti-CCNE1 (1:100, R&D Systems).

The following primary antibodies were used: anti-Akt (monoclonal rabbit, C67E7, #4691), anti-phospho (p)-Akt (S473, monoclonal rabbit, D9E, #4060), anti-Bcl-xL (monoclonal rabbit, 54H6, #2764), anti-Bcl-2 (polyclonal rabbit, #2876), anti-Cyclin D1 (monoclonal rabbit, 92G2, #2978), anti-CREB (monoclonal rabbit, 48H2, #9197), anti-p-CREB (S133, monoclonal rabbit, 87G3, #9198), anti-GAPDH (monoclonal rabbit, D16H11, #5174), anti-IGF-IR (monoclonal rabbit, D23H3, #9750 and monoclonal rabbit, D4O6W, #14,534), anti-phospho (p)-IGF-IR (Y1135/1136, monoclonal rabbit, 19H7, #3024), anti-PCNA (monoclonal rabbit, D3H8P, #13,110) and anti-Rb (polyclonal rabbit, #9313) (all purchased from Cell Signaling Technology). The SS18-SSX fusion protein was visualized using an anti-SS18/SYT antibody (#8819 Santa Cruz Biotechnology) detecting the N-terminus of SS18 (which is retained in the SS18-SSX fusion oncoprotein). Secondary antibody labeling (Bio-Rad) and immunoblot development were performed using an enhanced chemiluminescence detection kit (SignalFire ECL Reagent; Cell Signaling Technology) and a Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad), as described previously [14 (link), 40 (link)].

Protein extraction and Western blotting were performed according to standard procedures. Primary antibodies were used as follows: anti-c-MYC (1:1,000, Proteintech), anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4, anti-CDK6, anti-CCND1, anti-CCND2 and anti-CCND3 (1:1,000, Cell Signaling Technology) and anti-Actin (1:3,000, Sigma). The membrane was then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:3,000 Cell Signaling Technology). The intensity of protein bands was quantified by using the Image-J software package.

Cell extracts were prepared with mammalian protein extraction reagent (M‐PER; Thermo Fisher) plus protease inhibitor cocktail (Halt), and protein concentrations were determined by using the bicinchoninic acid assay (Thermo Fisher). Aliquots of protein lysates were separated on SDS‐10% polyacrylamide gels and transferred to polyvinylidene difluoride membrane filters, which were blocked with 5% blotting grade milk (Bio‐Rad, Hercules, CA, http://www.bio-rad.com) in TBST (20 mM Tris‐HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies, reacted with corresponding secondary antibodies, and detected by using a chemiluminescence assay (EMD Millipore, Billerica, MA, http://www.emdmillipore.com). Membranes were exposed to x‐ray film to visualize the bands (GE Healthcare Life Sciences, Piscataway, NJ, http://www.gelifesciences.com). The primary antibodies included anti‐p53, anti‐Rb, anti‐Oct4, anti‐SOX‐2, anti‐Nanog, anti‐c‐Myc, anti‐β‐catenin, and anti‐β‐actin (1:1,000; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com). The secondary antibodies included horseradish peroxidase‐conjugated donkey anti‐rabbit or anti‐mouse antibodies (1:2,000; GeneTex). The quantification of each protein expression level was normalized by internal control α‐tubulin, and the expression level of parental MSC was referred to as 1.

Antibodies against CDK5 (C-8), p35 (C-19), and TPX2 were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-β-actin, anti-GFP, anti-Rb, and pRb-Ser807/Ser811 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-ki67 antibodies and the CDK5 inhibitor roscovitine were acquired from Genetex and Cell Signaling Technology, respectively. TMX was purchased from Sigma. Lentivirus of CMV-EGFP-3FLAG-CDK5, CMV-EGFP-3FLAG-kinase-dead-CDK5, and control were obtained from Neuron Biotech (Shanghai). TPX2 was amplified by PCR and inserted into GFP-C3 to generate GFP-C3-TPX2 vector. GFP-TPX2-S486A and GFP-TPX2-S486D mutations were generated using a site-directed mutagenesis kit following the instruction manual (Stratagene).