Protein Extraction and Detection Protocols

Cells were lysed either by boiling in the SDS-containing loading buffer, or by vortexing with glass beads in case of immunoprecipitation (IP) experiments [31 (link)]. Protein were run on SDS-PAGE gel, or (in case of analysis of protein polymer), on semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) gel [97 (link),98 (link),99 (link)]. Positions of molecular weight markers for SDS-PAGE or SDD-AGE gels are shown on figures; it should be noted that the correspondence of the monomer positions to markers is grossly imprecise on SDD-AGE gels due to high diffusion. Anti-HA-agarose (Thermo Fisher Scientific, Waltman, MA, USA) was used for IP. The cycloheximide chase experiment was performed as described [31 (link)]. An equal number of cells was collected at the specific time point and lysed by boiling to isolate protein. Proteins in extracts or immunoprecipitates were detected by Western analysis followed by reaction to specific antibodies, such as: anti-HA HA.11 (Covance, Inc., Emerville, CA, USA); anti-Myc 9B11 (Cell Signaling Technology, Danvers, MA, USA); anti-Pgk (Molecular Probes, Inc., Eugene, OR, USA). In all experiments, we used appropriate secondary antibodies from GE Healthcare Chicago, IL, USA.