Anti actin ab8227

The whole-cell extracts were prepared and the proteins were resolved on a 4-12 % gradient polyacrylamide SDS-PAGE, as described previously [19 ]. The primary antibodies used were as follows: anti-RET (# 3220), anti-pAkt (# 9271), anti-Akt (# 9272) and anti-p-pRb (# 9307S) were purchased from Cell signaling, Beverly, MA, USA, anti-VEGF (sc-152), anti-Bcl-2 (sc-509), anti-c-Myc (product sc-40), anti-pERK1/2 (sc-81492), anti-ERK1/2 (sc-271270), anti-pMEK1/2 (sc-81503), anti-MEK1/2 (sc-81504), anti-E2F1 (sc-56661), anti-cyclin E (sc-247) and anti-pRb (sc-50) were purchased from Santa Cruz Biotechnology, and anti-actin (ab8227) was purchased from Abcam. Mouse or rabbit IgG antibodies conjugated with horseradish peroxidase (HRP) (BioRad) were used as secondary antibodies. An enhanced chemiluminescence kit (Santa Cruz Biotechnology) was used for the detection of the luminescence.

To confirm RNAi efficiency in RAW264.7 macrophages, cells were forward transfected with 20 μl of a solution with a final concentration of 1 μM of Silencer siRNA (Life Technologies) diluted in Opti-MEM (Life Technologies) mixed with 10 µl of 0.03% (vol/vol) Lipofectamine RNAiMax/Opti-MEM (Life Technologies) for 48 h (35 (link)). Total RNA from 1.6 × 10⁶ transfected cells (n = 3) was isolated and converted to cDNA, and quantitative reverse transcription-PCR (qRT-PCR) was performed using Sybr green (Life Technologies) (35 (link)). Relative expression was calculated using the ΔΔC_T method (73 (link)). For protein expression, 3 × 10⁵ transfected cells were harvested, and total lysates were analyzed by Western blotting using anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (PA1-988; Pierce), anti-Copβ1 (PA1-061; Pierce), and antiactin (ab8227; Abcam) antibodies as previously described (35 (link)). For expression of Rab-EGFP GTPases, RAW264.7 macrophages were transiently transfected with pEGFP-Rab11b and pEGFP-Rab4a (74 (link)) using JetPrime as described by the manufacturer (Polyplus).