Ez view red anti flag m2 affinity gel beads

Cells grown on 6-well plates were washed with PBS and solubilized at 4°C in 200 μl solubilization buffer (50 mM Tris-HCl pH 7.5, 1% Igepal, 1 mM EDTA, 0.1 mM PMSF, 10 mM iodoacetamide). Samples were centrifuged (21,000 g, 30 min). Suitable volumes of supernatant were mixed (2:1 ratio) with sample buffer (180 mM Tris-HCl, pH 6.8, 15% glycerol, 9% SDS, 0.075% Bromophenol Blue, 7.5% β-mercaptoethanol), electrophoresed and blotted as described [21 (link)]. Blots were probed with the required antibodies and stained with a chemiluminescent substrate (Amersham, Little Chalfont, UK). Comparable loading was ascertained by stripping and reprobing with an anti-ERK2 antibody. Stripping was performed by washing the membranes with PBS, followed by treatment with 0.5 N NaOH, 10 min at room temperature, and a final 10-min wash with H₂O.
For immunoprecipitation, cells were washed, solubilized and centrifuged as above. Supernatants were incubated with 20 μl of EZview Red ANTI-FLAG M2 Affinity Gel beads (Sigma) for 1 h at 4°C. The mixture was washed, centrifuged, eluted with pre-heated (95°C) sample buffer, and electrophoresed and blotted as above.

For immunoprecipitation, 500 μg/mL of LETM‐IgG (Patient 3) or ON‐IgG (Patient 17) was incubated with 5 μg of HEK 293T cell lysates with or without the overexpression of FLAG‐tagged GRP78 (Origene) for 4 h at 4°C, and then incubated with 40 μL of anti‐FLAG‐IgG coupling resin (EZview Red Anti‐FLAG M2 Affinity Gel beads; Sigma‐Aldrich), for 2 h at 4°C. After the GRP78 antigen–antibody complexes were precipitated, the supernatants (LETM‐IgG or ON‐IgG with/without GRP78 antibodies) were used for the analysis.10 Images were captured and evaluated by an In Cell Analyzer 2000 (GE Healthcare) at × 20 magnification.

Plasmids vectors containing HA and FLAG tags ligated to the C or N termini of the indicated ORFs were transfected into HEK293T cells as described below. After 18 hours of expression, cells were treated with lysis buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP-40), the extract was centrifuged for 10 minutes at 4°C, and the supernatant was removed. 25ul of washed EZ View Red Anti-FLAG M2 Affinity Gel beads (Sigma, St Louis MO, #F2426) were added to each extract and rotated overnight at 4°C. Extract was then washed 3 times with lysis buffer and re-suspended in SDS PAGE loading buffer before boiling and electrophoresis.
For ubiquitination or deubiquitination experiments, the protocol above was followed with the addition of HA-tagged ubiquitin (HA-Ub) or mutant ubiquitin plasmids. Mutant ubiquitin plasmids that are only able to be ubiquitinated at either K48 or K63 were added to the transfection experiments as described above. The mutant and wildtype ubiquitin plasmids were previously described [35 (link)].

Cells were transfected with the following plasmids: UAS-Flag-Mad, UAS-Mad-AVA, pAC-Dullard or pAWF-Tkv-Flag. All cell samples were lysed in RIPA buffer containing phosphatase and protease inhibitors. EZview Red anti-Flag M2 Affinity gel beads (Sigma) were used following standard protocols. All subsequent western blotting was carried out using standard protocols. Primary antibodies were used at the following concentrations, anti-Flag (Sigma), 1:1000; anti-Dullard (Sigma), 1:1000. Secondary antibodies (Thermo Scientific) anti-mouse and anti-rabbit, 1:1000.