HDAC6 Regulates EGFR Trafficking Figure 9. Treatment with nocodazole decreased EGFR intracellular transport. (A) Pkd1 wild type MEK cells were serum-starved and preteated with nocodazole (0.2 mg/ml) for 30 min. The pretreated cells were further treated with nocodazole and 80 ng/ml EGF-Alexa Fluor 488 for indicated periods of time. Then, cells were stained with anti-EEA1 antibody. Localization of EGF-Alexa Fluor 488 to the EEA1-containing vesicles was analyzed. * p , 0.05. (B) Pkd1 mutant (Null) MEK cells were treated, processed for immunostaining, and analyzed as in (A). * p , 0.05. (C) Pkd1 wild type MEK cells were treated as in (A) and then processed for immunostaining with anti-LAMP2 antibodies. Co-localization of EGF-Alexa Fluor 488 to LAMP-2-containing vesicles was examined. * p , 0.05. (D) Pkd1 mutant (Null) MEK cells were treated, processed for immunostaining with anti-LAMP2 antibodies, and analyzed as in (C). * p , 0.05. doi:10.1371/journal.pone.0049418.g009 Studies Hybridoma Bank); anti-acetylated-a-tubulin, anti-actin antibody (Sigma, MO). Donkey-anti-rabbit, donkey-anti-mouse, donkey-anti-goat IgG-horseradish peroxidase (Santa Cruz, CA) were used as secondary antibodies for Western blotting. EGF- Alexa Fluor 488 was purchased from Invitrogen (Invitrogen, CA). Fluro488-conjugated anti-mouse and anti-rabbit IgG antibody, Fluro555-conjugated anti-mouse, anti-rabbit, anti-goat, anti-rat IgG antibody (Invitrogen, CA) were used as secondary antibodies for immunofluorescence. Immunofluorescence images were ob- tained with a NIKON ECLIPSE 80i Microscope. For saponin treatment, cells were first rinsed with cold PBS three times and incubated with 0.005% (w/v) saponin that was made freshly in 4 uC PBS. Saponin treatment lasted 5 min on ice and was stopped by quickly rinsing the cells three times with cold PBS. Then regular immunostaining procedures were applied. For EGF treatment, cells were serum-starved overnight and then treated with EGF (100 ng/ml) or EGF-Alexa Fluor 488 (80 ng/ml). HDAC6 Knockdown by Lentivirus Carrying HDAC6 shRNA HEK293T cells were cotransfected with lentiviral plasmid pGIPZ-siHDAC6 or control empty vector pGIPZ-NS, psPAX2 packaging plasmid, and pMD2.G envelope plasmid using calcium phosphate. Twelve hours later, medium containing the trans- fection reagent was removed and replaced with fresh complete DMEM medium plus 10% FBS and penicillin/streptomycin. Forty-eight hours later, cultures containing lentiviral particles were harvested from HEK293T cells. Pkd1 wild type and mutant MEK cells and PN24 cells were then infected with appropriate amounts of lentiviral particles together with 5 mg/ml polybrene (Sigma, MO). Twenty-four hours later, virus-containing medium was removed and replaced with fresh medium plus 5 mg/ml puromy- cin. Two days after selection, all the cells are GFP positive, which indicates the efficiency of transduction. Five days after infection, cells were harvested and analyzed by Western blot to examine the efficiency of HDAC6 knockdown. Quantitative Reverse-transcription Polymerase Chain Reaction HADC6 was stable knockdown in Pkd1 mutant (Null) MEK cells. Cells were washed quickly with PBS, and total RNA was extracted using the RNeasy plus mini kit (Qiagen Germanton, MD). One micrograms of total RNA were used for reverse transcription reactions in a 20 ml reaction to synthesize cDNA using Iscript cDNA synthesis kit (BioRad, Hercules, CA). RNA expression profile was analyzed by real-time PCR using iTaq SYBER Green supermix with ROX (BioRad) and carried in an PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49418