P akt ser473 and thr308

Cells were seeded in 12-well plates at a density of 200,000 cells/well. The next day, the culture medium was replaced with media containing DMSO, 50 nM dabrafenib, 2.5 μM AKTi or the combination. After 24 hours, the media was removed and the cells were lysed directly in the wells for 15-20 min with lysis buffer (Pierce RIPA buffer, Thermo Scientific) containing phosphatase- and protease inhibitors (Sigma) and protein was extracted for western blot analysis as previously described [19 (link)]. Blots were blocked and probed with primary antibodies in 5% milk or 5% bovine serum albumin (BSA) in 1% Tween-20 phosphate-buffered saline (PBS-tween), washed with PBS-tween three times and incubated with horse-radish-linked secondary antibodies. Primary antibodies included p-AKT Ser473 and Thr308, AKT, p-S6K Thr389, S6K, p-S6 Ser235/236, S6, p-4EBP-1, 4EBP-1, p-GSK-3β, GSK-3β and GAPDH (all from Cell Signaling Technology, Danvers, MA). The immunoreactivity was visualized by use of an ECL-2 kit (Pierce, Thermo Scientific) and scanning of the blots by a Typhoon scanner (Amersham Biosciences Co, Piscataway, NJ). Quantification of protein levels from western blot analysis was done using ImageQuant software (version 5.2 Molecular dynamics).

The following antibodies were used for immunoassays: p‐S6 Ser240/244 (Cell Signalling, 5364 and 6520), S6 (Cell Signalling, 2217), p‐4EBP1 Thr37/46 (Cell Signalling, 2855), 4EBP1 (Cell Signalling, 9644), p‐AKT Ser473 and Thr308 (Cell Signalling, 9271 and 9275), AKT (Cell Signalling, 4691), p‐STAT3 Tyr705 (Cell Signalling, 4113 and BioLegend, 651021), STAT3 (Cell Signalling, 9139), p‐P65 (Cell Signalling, 3031 and 5733), P65 (Cell Signalling, 8242), Vinculin (Sigma, V4505), Iba1 (Wako, 019‐19741), CD11b (eBiosciences, 17‐0112‐82) and (Abcam, ab8878), CD45 (BioLegend, 103105), Ki67 (BioLegend, 652413), Granzyme b (BioLegend, 337221) and Perforin (BioLegend, 154406). IFNʏ (BioLegend, 505826), CD49d (BioLegend, 103618 and 304313), CD44 (BioLegend, 10349), CD62L (BioLegend, 104438), CD3 (BioLegend, 152303), CD4 (BioLegend, 100427), CD8 (BioLegend, 100722 and 100732), PU.1 (Cell Signalling, 2258), c‐kit (Abcam, ab212518), CD41 (BioLegend, 303702), CD235a (Life Technology, 14‐9987‐82), P2RY12 (Atlas, HPA014518 and 848006), TREM2 (Abcam, ab86491),TMEM119 (Sigma, HPA051870), MHCII (BioLegend, 107607), F480 (BioLegend, 123130), Sox2 (Santa cruz, sc‐17320) and NANOG (BioLegend 16H3A38).