Rabbit anti hdac6

Immunofluorescence was performed on CD41⁺ cells cultured with or without inhibitors^{4 (link)}. In brief, cells were allowed to adhere on poly-l-lysine-coated slides for 1 h at 37 °C, fixed in 4% PFA, permeabilized with 0.2% of Triton, and blocked with 1% BSA before antibody labeling. Cells were examined under a Leica-SpE confocal microscopy with ×63/1.4NA oil objective. The following antibodies were used: mouse anti-ac-tubulin (1:300, Sigma T7451), mouse anti-Tubulin (1:100, Sigma T5293), rabbit anti-HDAC6 (1:100, Cell Signaling 7558), and rabbit anti-Cortactin (1:100, Millipore 05-180). FITC-conjugated phalloidin (1:500, Sigma P5282) was used to stain actin cytoskeleton, mouse anti-CD63 (1:100, Sigma SAB4700215, clone MEM 259), and rabbit anti-VWF (1:1000, Dako A0082). Appropriate secondary antibodies conjugated with Alexa 488, Alexa 546, or Alexa 633 (Molecular Probes) were used. DAPI (Molecular Probes) was applied for nucleus staining. Three-dimensional image analyses were performed with Zeiss Image Examiner software.

Primary antibodies used for immunocytochemistry (ICC) and WB included mouse anti-aaTub (1:10,000; Sigma, St. Louis, MO, USA; Cat #T6793), rabbit anti-ARL13B antibody (1:5000; Proteintech, Rosemont, IL, USA; Cat #17711-1-AP), mouse anti-TUJ1 (1:2000; R&D Systems, Minneapolis, MN, USA; Cat #NL1195V), chicken anti-Ki-67 (1:5000; Encor, Gainesville, FL, USA; Cat# CPCA-Ki67), chicken anti-GFAP (1:1000; Encor, Gainesville, FL, USA; Cat# CPCA-GFAP), mouse anti-GAPDH (1:10,000; Encor, Gainesville, FL, USA; Cat# MCA-1D4), chicken anti-GFP (1:5000; Abcam, Cambridge, UK; #ab13970), rabbit anti-HDAC6 (1:1000; Sigma, St. Louis, MO, USA; Cat #H2287), rabbit anti-HDAC6 (1:1000; Cell Signaling, Danvers, MA, USA; Cat #7558S), mouse anti-KIF3A (BD Biosciences, San Jose, CA, USA; Cat #611508), and rabbit anti-PCM1 (1:1000; Bethyl Laboratories, Montgomery, TX, USA; Cat# A301-150A). ACY-1215 (APEXbio, Houston, TX, USA; Cat #A4083) and ACY-738 (MedChemExpress, Monmouth Junction, NJ, USA; Cat #1375465-91-0) were dissolved in 100% DMSO (Fisher Scientific, Waltham, MA, USA; Cat # D128-500) to produce a stock concentration of 10 mM. cDNA vectors pCMV-EGFP or pCMV-mycEGFP:HDAC6[#NM_001321225.2] were designed and obtained from Vectorbuilder (Vectorbuilder.com (accessed on 29 March 2021)).

The cells were lysed in radio-immunoprecipitation assay (RIPA) buffer. Protein samples were separated insodium dodecyl sulfate–polyacrylamide gel electrophoresis, thereafter transferred into polyvinylidenefluoride (PVDF) membrane. The membranes were probed with the primary antibodies overnight at 4 °C, followed by anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1000) for 1 h. Protein bands were visualized by using enhanced chemiluminescence assay kit and quantified using Image J software.The following primary antibodies were used in present work: rabbit anti-HDAC6 (1:1000) (#7558), rabbit anti-phospho-AKT (1:1000) (#4058), rabbit anti-AKT pan (1:1000) (#4685), rabbit anti-phospho-mTOR (1:1000) (#2971), rabbit anti-mTOR (1:1000) (#2983), mouse anti-PCNA (1:1000) (#2586), mouse anti-GAPDH (1:10,000) (#97,166), and rabbit anti-Tubulin (1:1000) (#2146) (Cell Signaling Technology, Denvers, MA). The phospho-AKT antibody we used is phospho-Akt (Ser473) which could recognize endogenous AKT1, 2, and 3 only after phosphorylation at ser473 site. The relative intensity of phospho-AKT was calculated by phospho-AKT versus total AKT and so was the relative intensity of phospho-mTOR. The relative intensity of HDAC6, AKT, mTOR, and PCNA protein was calculated by corresponding protein versus internal control such as GAPDH or ß-Tubulin, respectively.