Immunofluorescence Microscopy of ASK1 and EB1

Primary antibodies directed against ASK1 (Abcam), EB1 (BD Biosciences), α-tubulin (Abcam), γ-tubulin (Abcam), phosphoserine (Cell Signaling Technology), phosphothreonine (Cell Signaling Technology), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz), rhodamine- or fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch) were obtained from the indicated sources. The pT206-EB1 customized antibody was obtained from GL. DAPI, and fluorescein-conjugated phalloidin were purchased from Sigma-Aldrich. The control siRNA (5′-CGUACGCGGAAUACUUCGA-3′) and ASK1 siRNAs (#1: 5′-GCACUCCU-UCAUCGAGCU-3′; #2: 5′-GGUAUACAUGAGUGGAAUU-3′) were synthesized by Ribo Bio. Mammalian expression plasmids for GFP-EB1 were constructed by PCR using the pEGFP-N1 vector as described previously [31 (link)].