P44 42 mapk erk1 2 137f5

Fibroblasts were collected and suspended in RIPA buffer (Radio-Immuno Precipitation Assay Buffer). Forty micrograms of total protein were analyzed by 10% denaturing polyacrylamide gels and then transferred electrophoretically to PVDF membranes (Immobilon-NC, Millipore, Italy). Anti-A2BR (H-40) (Santa Cruz Biotechnology), anti HIF1α (A300-286A) (Bethyl Laboratories, Tema Ricerca, Italy) or anti CD73 (5NT5E, C-terminal) (Sigma-Aldrich, Milan, Italy) or phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) or p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling Technology) primary antibodies were used. Immunoreactive protein bands were visualized by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK) and analyzed to Las4000 (GE Healthcare Life Sciences).

For direct IB analysis, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with phosphatase inhibitors. The following primary antibodies were used against: cleaved caspase-3 (Asp175) (5A1E) (#9664), cleaved caspase-8 (Asp391) (18C8) (#9496), RB (#9309), p-RB (Ser870/811) (#8516), CDK2 (#2546), p-CDK2 (#2561), Bcl-2 (#15071), Bax (#5023), p21 (#2947), p27 (#3686), cyclin D1 (#55506), cyclin E (#4136), p44/42 MAPK (Erk1/2) (137F5) (#4695), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (#4370), Akt (pan) (40D4) (#2920), phospho-Akt (Ser473) (D9E) XP (#4060), PI3 kinase p110α (C73F8) (#4249), cyclin A2 (#91500), and cyclin B1 ( #4135), all purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to CDK4 (#11026-1-AP), CDK6 (#14052-1-AP), SKP2 (#15010-1-AP), p53 (1C12) (#2524), P16-INK4A (#10883-1-AP), EGFR (#18986-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#60004-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). The anti-FLAG was obtained from Sigma-Aldrich (St. Louis, MO, USA). All secondary antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.

Antibodies against mouse cell markers and the isotype controls used in flow cytometry assay were as follows: BAFFR-ATTO 488 (9B9) and rat IgG2b-ATTO 488 (A-1) from Enzo Life Sciences, Inc (Farmingdale, NY); BAFFR-FITC (clone 7H22-E16), rat IgG1-FITC and fixable viability dye eFlour 780 from (eBioscience, San Diego, CA); CD138-PE (281–2), rat IgG2a-PE (R35-95), and BV605-IgD (11-26C.2a) and BV605-rat IgG2a,λ (B39-4) from BD Biosciences/BD Pharmingen (San Jose, CA); PerCP/Cy5.5-IgM (RMM-1), PerCP Cy5.5 Rat IgG2a,κ (RTK2758), Pacific Blue-CD19 (6D5), Pacific Blue Rat IgG2a,κ (RTK2758), CD93-APC (AA4.1), rat IgG2b-APC (RTK4530) from BioLegend (San Diego, CA); and Annexin V-Alexa Fluor 647 (Invitrogen, Grand Island, NY). Propidium iodide (0.5 μg/ml) was used to identify dead cells (BD Biosciences/BD Pharmingen). B cell isolation Kits were purchased from Miltenyi Biotec Inc (San Diego, CA) to negatively select B cells from spleen as described previously [33 (link), 34 (link)]. BAFF was purchased from R&D Systems. Antibodies used in Western blot analysis were as follows: NF-κB2 p100/p52, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) XP-HRP, p44/42 MAPK (ERK1/2) (137F5), and β-actin-HRP (13E5) (Cell Signaling Technologies, Danvers, MA).