Hrp conjugated goat anti rabbit or anti mouse antibody

HEK 293T cells were transfected in the presence of Lipofectamine 2000 (Invitrogen). One of the following combinations of constructs were used for transfection: (1) a cDNA encoding the GFP-tagged myosin IIIa tail alone (aa 1,107–1,621; accession number NM_148413); (2) a cDNA encoding the GFP-tagged myosin IIIa tail with a cDNA encoding the Flag-tagged MORN4 fragment (aa 1–146; accession number NM_198108); or (3) a cDNA encoding the GFP-tagged myosin IIIa tail with a cDNA encoding the Flag-tagged spectrin βII-R16 fragment, used as a negative control (C-terminal region of spectrin βII starting from the spectrin repeat 16; aa 1,914–2,364; accession number NP_008877.1). Protein extracts were incubated at 4°C for 2 h with a mouse monoclonal antibody directed against FlagM2 (Sigma-Aldrich) and then for 30 min with protein G–coated µMACS magnetic MicroBeads (Miltenyi Biotec). The bound proteins were denatured and eluted in 50 µl 2× NuPAGE containing 1× SDS. Immunoprecipitates were run on 4%–12% NuPAGE gels (Invitrogen) and subjected to Western blot analysis. HRP-conjugated goat anti–rabbit or anti–mouse antibodies (Jackson ImmunoResearch Laboratories) and the ECL chemiluminescence system (Pierce) were used for detection of the proteins.

For Western blotting, yeast cultures were grown at 30°C in 3 ml of either SD plus glucose or SD plus galactose/raffinose and harvested. Equal numbers of cells were pelleted and resuspended in 0.2 ml of 0.1 M NaOH and incubated for 5 min at room temperature as described (Kushnirov, 2000 (link)). After centrifugation, the resulting cell pellet was resuspended in sample buffer and heated to ∼100°C for 3 min. Lysates were separated on a 10% SDS polyacrylamide gel and electroblotted to PVDF in 25 mM Tris and 192 mM glycine supplemented with 0.05% SDS and 10% methanol for 30 min. Rabbit anti–c-Myc polyclonal (GenScript) or anti–α-tubulin (Applied Biological Materials, Inc.) monoclonal antibodies and HRP-conjugated goat anti–rabbit or anti–mouse antibody (Jackson ImmunoResearch Laboratories) were used at 1:1,000, 1:1,000, 1:3,000, or 1:3,000, respectively. The chemiluminescence signal was acquired on an ImageQuant LAS 500 gel documentation system. Immunoblots were exposed without saturating the camera’s pixels.