Anti akt 40d4

Cells were resuspended to a concentration of 2 million/ml in RPMI with 0.2% FBS. We stimulated cells with 10 nM fMLP and quenched the reaction at the indicated time points by adding aliquots of the cell mixture to ice-cold 20% trichloroacetic acid (TCA) containing the phosphatase inhibitors 40 mM NaF and 20 mM β-glycerol phosphate (50020; Fluka, St. Gallen, Switzerland). The samples were spun at 20,000 × g for 15 min to pellet. The sample pellets were washed with 0.5% TCA and resuspended in Laemmli protein sample buffer (161-0737; Bio-Rad) containing 5% β-mercaptoethanol. Protein bands were separated by SDS–PAGE gel electrophoresis, transferred to nitrocellulose, blocked with Odyssey block, and incubated at 4°C overnight with 1:1000 dilutions of anti–phospho-PAK (2605S; Cell Signaling, Danvers, MA) and anti-Pak2 (4825S; Cell Signaling) or anti–phospho-Akt (4060S; Cell Signaling) and anti-Akt (40D4; Cell Signaling). The blot was developed with the fluorescent secondary antibodies, and protein bands were imaged using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Whole‐cell lysates for Western blot analyses were obtained by the direct lysis of cells in 2× Laemmli buffer at 90°C. All samples were resolved by SDS‐PAGE, transferred to PVDF membranes (1620177; Bio‐Rad, Hercules, CA, USA) and probed with the appropriate antibodies. To study the TIF by immunostaining, cells that were plated on glass coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, and incubated with primary and secondary antibodies.23The following antibodies were used for Western blotting: anti‐RhoGDIα (133248; Abcam, Cambridge, England), anti‐TRF1 (GTX70290; Genetex, Irvine, CA, USA), anti‐TRF2 (OP129; Calbiochem, Darmstadt, Germany), anti‐β‐actin (sc‐47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐GAPDH (10494‐1‐AP; Proteintech Group, Chicago, IL, USA), anti‐Akt (40D4; Cell Signaling Technology), and anti‐phospho‐Akt (Ser473) (9271; Cell Signaling Technology, Danvers, MA, USA). For immunostaining, the antibodies included anti‐TRF2 (OP129; Calbiochem), and anti‐53BP1 (NB100‐304; Novus Biologicals, Littleton, CO, USA).