Anti pten 138g6

Sixty milliliters of CM was prepared with 1 × 10⁶ per ml of BMDMs that had been stimulated with nonapoptotic or apoptotic 344SQ cells. In the case of unconventional secretion of PTEN, the medium was diluted 1:1 with 2× exosome lysis buffer (4% SDS, 2% Triton-X100, 0.1 M Tris pH 7.4 and 2× protease inhibitors) and lysed for 1 h at 4 °C.^{61 (link),62} The resulting medium-lysis buffer mixture was filtered through a 0.22-micron filter (Macherey-Nagel) and divided into 15-ml vials. Twenty-five microliters of anti-PTEN (138G6, Cell Signaling) was added to a mixture vial and incubated for 4 h with rotation at 4 °C. Immunocomplexes were then precipitated with 100 μl (50% slurry) of Protein A/G Sepharose (BioVision Inc.). For the remaining mixture vials, the pull-down beads with the prebound immunocomplexes were added to new tubes and incubated for 4 h with rotation at 4 °C repeatedly. They were then washed four times with IP wash buffer (25 mM HEPES pH 7.4, 1 M NaCl, 1 mM EDTA, 0.5% Triton X-100). To avoid overlap with IgG heavy chains, western blotting for PTEN immunoprecipitates was performed in nonreducing conditions.

Human wild type (Wt), N-terminal myristoylated (myr)-tagged, and C-terminal NLS-tagged PTEN complementary DNA (cDNA) sequences were subcloned from pCDNA3-PTEN into pTRIPZ vector (Addgene, Watertown, MA, USA) with AgeI and MluI to generate PTEN expression plasmid. All plasmid constructs were confirmed by sequencing. Ampicillin, puromycin, and doxycycline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA, USA), while RPMI, DMEM, Opti-MEM reduced serum media, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Protein G Sepharose 4 Fast Flow beads were purchased from Cytiva (Malborough, MA, USA). The antibodies for Western blotting were as follows: anti-PTEN (138G6, 1:1000), anti-p-AKT (9271, 1:1000), anti-AKT (9272, 1:1000), anti-GAPDH (D16H11, 1:4000), anti-Lamin B1 (D4Q4Z, 1:1000), anti-β-Tubulin (9F3, 1:1000), and anti-EGFR (D3871, 1:1000) antibodies, all purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-HSP90 (610418, 1:4000) was purchased from BD Bioscience (Franklin Lakes, NJ, USA).

For Western blot, cell and tissue lysates were prepared with 150-RIPA buffer and protease and phosphatase inhibitor cocktails (Roche). The following antibodies were used for Western blotting: rabbit polyclonal anti-PTEN (138G6; 1:1000, Cell Signaling Technology), rabbit polyclonal anti-phospho-Akt(S473) (9271S; 1:1000, Cell Signaling Technology), rabbit polyclonal anti-Akt (9272S; 1:1000, Cell Signaling Technology), rabbit polyclonal anti-GAPDH (14C10; 1:6000, Cell Signaling Technology), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (9102S; 1:3000, Cell Signaling), rabbit polyclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101S; 1:3000, Cell Signaling).