Phospho s144 141 pak1 2

Proteins from cell lysates were separated using 15% polyacrylamide gels und transferred onto nitrocellulose for 2 h at 120 V, followed by blocking with 5% (w/v) nonfat dried milk for 1 h. Primary antibodies were incubated over night at 4°C with dilution according to the manufacturers´ instructions ((beta-actin, Mab AC-40, Sigma-Aldrich, St. Louis, MO, USA; dilution 1:5,000); PAK2 (Cell signaling 2608, dilution 1:1,000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:1,000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1,000); Rac1 (Millipore 05-389, clone 23A8; dilution 1:1,000); LRP1 (Abcam ab92544; dilution 1:50,000); TfR (Invitrogen 13-6,800, clone H68.4; dilution 1:1,000); gp96 (R&D Systems 816803, dilution 1:1,000) in TBST buffer (50 mM Tris-HCL, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w/v) Tween 20) and subsequently for 1 h at room temperature with HRP-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:5,000; rabbit Rockland 611-1302; dilution 1:5,000). For the chemiluminescence reaction, ECL Femto (Fisher Scientific, Schwerte, Germany) was used. The signals were detected with the INTAS Chemo Cam Imager (Intas Science Imaging Instruments GmbH, Göttingen, Germany) and analyzed densitometrically using the LabImage 1D software (Kapelan Bio-Imaging GmbH, Leipzig, Germany).

Cells lysates were separated on 15% polyacrylamide gels and transferred onto nitrocellulose for 2 h at 250 mA, followed by blocking with 5% (w/v) nonfat dried milk for 1 h. Blots were incubated with the appropriate primary antibody with dilution according to the manufacturers’ instructions (beta-actin, Mab AC-40, Sigma-Aldrich, St. Louis, MO, USA; dilution 1:5000); MAPKAPK-2 (Cell signaling 3042, dilution 1:1000); pT222-MAPKAPK-2 (Cell signaling 3316, dilution 1:1000); PAK2 (Cell signaling 2608, dilution 1:1000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:2000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1000); Rac1(Millipore 05-389, clone 23A8; dilution 1:1000) in buffer B (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w/v) Tween 20) for 18 h and subsequently for 2 h with a horseradish peroxidase-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:3000; rabbit Rockland 611-1302; dilution 1:3000). For the chemiluminescence reaction, ECL Femto (Fisher Scientific, Schwerte, Germany) was used. The signals were analyzed densitometrically using the KODAK 1D software (2004, Rochester, MN, USA).