Anti ha

The cells were collected and protein extraction was carried out as described in [32 (link)]. Briefly, approximately ~10 OD₆₀₀ cells were collected by centrifugation and total protein was precipitated and extracted using 10% trichloroacetic acid and resuspended in SDS-glycerol buffer. The extracted proteins were estimated by BCA (bicinchoninic acid) protein assay kit. The samples were normalized for protein amounts and an equal amount of protein from all the samples were run on 4–12% bis-tris gels (Invitrogen, NP0336BOX). For the Far11 mobility shift assay, 7% SDS-PAGE gel was used. The relevant portion of gel was cut and used for transfer and blotting. The lower portion was stained with Coomassie blue for protein loading normalization. The blots were developed using the following antibodies: anti-FLAG raised in mouse (1:2000; Sigma-Aldrich, F1804), anti-HA raised in mouse (1:2000; Sigma-Aldrich, 11583816001), anti-HA raised in rabbit (1:2000; Sigma-Aldrich, H6908), anti-mouse horseradish peroxidase (HRP)-conjugated antibody (1:4000; Cell Signaling Technology, 7076S), anti-rabbit HRP-conjugated antibody (1:4000; Cell Signaling Technology, 7074S). For chemiluminescence detection, western bright ECL HRP substrate (Advansta, K12045) was used. Alkaline phosphatase treatment was performed as described in [32 (link),57 (link)].

Histones were isolated from log phase of WT strains and mutants using total histone extraction kit (Epigentek, USA), then separated by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to PVDF membranes (110 V, 3.5 h). The membranes were blocked in 5% nonfat milk and incubated with anti-HA (1:300, Millipore) primary antibody overnight at 4°C. Following three washes, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:800, Millipore) for 1 h at 37°C. Finally, blots were developed with Western Blotting Detection Reagent (Engreen, China) according to the product instructions.

Yeast proteins were harvested by glass beads grinding in PBS buffer with vigorous vortex. Total proteins were subjected to electrophoresis in SDS-PAGE gel and transferred to nitrocellulose membrane. Anti-HA (Cat. 05904, Millipore, U.S.A.) was used to detect Dnm1-HA. Relative Dnm1 levels were determined by Odyssey Infrared Imaging Systems (LI-COR Bioscience, U.S.A.), and normalized based on actin levels.

Embryos were fixed in 2% paraformaldehyde in seawater for 1 hr then washed 4 times in PBST, 10 min each. Embryos were incubated in PBST + 5% goat serum for 1 hr at room temperature, or overnight at 4°C, with the primary antibody diluted 1:1000 (anti-GFP, anti-myc, anti-RFP; Invitrogen, Grand Island, NY), 1:650 (anti-HA; Millipore, Billerica, MA), 1:300 (anti-PKC ζ; Santa Cruz Biotechnology, Dallas, TX), or 1:250 (anti-phospho-Myosin Light Chain 2 [Ser19]; Cell Signaling Technology, Danvers, MA). Animals were washed 5× for 10 min in PBST and then placed in an appropriate secondary antibody, and incubated as described for the primary antibody. Secondary antibodies used were anti-mouse or anti-rabbit Alexa Fluor-labeled antibodies (Invitrogen) with a range of excitation/emission spectra, depending on the experiment. Samples were washed 4 to 10× in PBST following secondary incubation. For microscopy, labeled embryos were immobilized onto cover slips coated with 0.08% Poly-L-lysine. Fixed embryos were cleared in 80% glycerol or in an isopropyl alcohol series followed by 2:1 benzyl alcohol:benzoyl benzoate (BABB).