Anti ha agarose

Expression vectors encoding HA-MKK7, flag-septin 11-WT (wild-type), and c-Myc-GADD45β (GenScript Inc.) driven by CMV were transfected into HEK-293FT cells. To test the requirement for septin 11-MKK7 interaction in the role of septin 11 in cellular responses to 27HC, flag-septin 11-∆G4 and flag-septin 11-∆GTP were also generated. All constructs were confirmed by sequencing. Seventy-two hours post-transfection, the cells were pelleted and lysed, and proteins were extracted using a buffer containing 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, and protease inhibitors. The recombinant proteins were purified using anti-HA agarose (Thermo Fisher Scientific, 26181), anti-flag M2 affinity gel (Sigma, A2220), or anti-c-Myc agarose beads (ThermoFisher Scientific, 20168), and elution with 0.1 M glycine, pH3.5, followed by neutralization to pH 7.0. Proteins were freshly prepared prior to use. In pull-down assays the beads were pre-blocked with 1% BSA to reduce nonspecific binding, and the bait protein was added and incubated with beads for 1 h at 37 °C. After extensive washing the prey protein(s) was added and incubation resumed at 37 °C for 1 h. Following washing X3, protein complexes were eluted and samples were subjected to SDS-PAGE and immunoblot analysis.

A previously described procedure of protein extraction buffer was used to extract protein from pepper leaves [3 (link)]. The extracted protein was incubated overnight with anti-HA agarose at 4 °C (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic crack was used to collect beads and washed thrice with Tris buffer saline (TBS) and tween 20 (0.05%). Eluted protein was observed using immuno-blotting and by anti-HA-peroxidase (Abcam, Cambridge, UK).

For identification of ubiquitination sites on GSK3β, HEK293T cells were transfected with HA-tagged GSK3β expression plasmids for 36 h. Cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.4, 1% SDS), boiled for 5 min, sonicated and then diluted 10-fold with NP-40 lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40). After centrifugation at 15,000g for 15 min, the lysates were incubated with 120 μl of anti-HA Agarose (Thermo scientific) at 4 °C overnight with rotation. The beads were washed four times with NP-40 lysis buffer and eluted with 2 × SDS–PAGE sample buffer without a reducing agent. HA-GSK3β proteins eluted from the beads were subjected to SDS–PAGE followed by Coomassie Blue staining or western blot analysis. For MS analysis, gel bands from the Coomassie Blue stained-gel were excised and subjected to trypsin digestion and liquid chromatography–MS/MS. MS/MS data were analysed using SEQUEST (Thermo Finnigan, San Jose, CA) software to identify ubiquitin modification with the GG or LRGG remnant tag on lysine residues of GSK3β.

Total protein extracts were incubated with anti-HA agarose (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C. Beads were collected and washed with Tris-buffered saline and Tween-20 (0.05%). Eluted proteins were analyzed by immune-blotting using an anti-HA–peroxidase antibody (Abcam, Cambridge, UK).

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.