Ab109553

The activities of RhoA, Rac1 and Cdc42 from the cortex and hippocampus were measured using RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (BK030, Cytoskeleton). In brief, tissues were ground in liquid nitrogen and incubated in a lysis buffer 50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 10 mM MgCl₂, 2% Igepal and protease inhibitors with rocking at 4°C for 1 h. Sample lysates were centrifuged for 30 min at 16,000 g at 4°C and supernatants were collected into new tubes. The BCA method was used for quantitation of total protein concentration. 400 μg total protein was incubated with 50 μg rhotekin-RBD beads (for RhoA activation assay) or 10 μg PAK-PBD beads (for Rac1/Cdc42 activation assay) on a rotator at 4°C for 1 h. Then the beads were pelleted by centrifugation at 5,000 g for 1 min at 4°C and the supernatant was removed. After washing with a wash buffer, the beads were resuspended and centrifuged to remove the supernatant. The bound proteins were suspended in 20 μl 2× Laemmli sample buffer and boiled for 2 min. The sample was separated in 15% SDS-PAGE for the western blot analysis with the following primary antibodies: RhoA(1:1500, ab68826, Abcam), Rac1(1:1500,ab33186, Abcam), Cda42(1:1500, ab109553, Abcam).

HeLa cells were transfected with siRNA using Lipofectamine 2000 for 2×48 h or 1×72 h according to the manufactures’ recommendations. The siRNAs used were human-specific stealth siRNA (Invitrogen) for siRNA ctrl (negative control medium GC duplex), caveolin1 (HSS141467), Clathrin HC (HSS102017), GRAF1 (HSS118163) and cdc42 On target plus siRNA (J-005057-05) (Dharmacon). The cells were seeded on coverslips 24 h before optimal knockdown for PI assay. The efficiency of siRNA-based knockdown of endocytic proteins was determined by western blot. Primary antibodies used were mouse anti-GAPDH 1:5000 (MAB374, Millipore), rabbit anti-caveolin1 1:10,000 (ab2910, Abcam), mouse anti-Clathrin HC 1:1000 (clone 23 610499, BD Biosciences), rabbit anti-GRAF1 (Ra83) (Lundmark et al., 2008 (link)) and rabbit anti-cdc42 1:1000 (ab109553, Abcam). Secondary antibodies, donkey anti-mouse and donkey anti-rabbit IgG coupled to IRDye 680LT or 800CW (Li-Cor Biosciences) were used for fluorescent detection using an Odyssey Sa instrument (Li-Cor Biosciences). For detection with ECL Prime (GE Healthcare) anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase (HRP) from Agrisera or Sigma-Aldrich were used. The level of knockdown was quantified using Image Studio (Li-Cor Biosciences) for fluorescent images and ImageJ for films (Schneider et al., 2012 (link)).