Mouse anti ubiquitin p4d1

The following antibodies were used for detection
of endogenous and overexpressed proteins by immunoblots: mouse anti-Ubiquitin
(P4D1, Santa Cruz Biotechnology, 1:1000 dilution), mouse anti-HA (16B12,
Enzo Lifesciences, 1:1000 dilution), rabbit anti-mGFP^{47 (link)} (self-made, 1:1000 dilution), mouse anti-GAPDH (1D4, Enzo
Lifesciences, 1:1000 dilution), or mouse anti-β-actin (clone
AC-15, Sigma-Aldrich, 1:5000 dilution).

Native samples, freshly prepared, were diluted in native gel sample buffer (240 mM Tris-HCl, pH 8.8, containing 80% glycerol and 0.04% (m/v) bromophenol blue). Eight micrograms of total protein per lane were applied in Tris-HCl 4–15% gradient gels (Ready Gel, Bio-Rad). The native PAGE was run in an ice box at 16 mA, until the bromophenol blue dye reached the edge of the glass plate in 25 mM Tris-HCl, 192 mM glycine, pH 8.3. The gels were incubated for 15 min in transfer buffer (25 mM Tris, 192 mM glycine, containing 0.1% (m/v) SDS, and 20% (v/v) methanol) containing additionally 1% (m/v) SDS. The proteins were blotted at 0.8 mA/cm² for 2 h. For SDS-PAGE, the protein samples were boiled for 3 min in the presence of 2% SDS and 0.1 M 2-mercaptoethanol, then subjected to 12% SDS-PAGE, and thereafter transferred onto a PVDF membrane (Millipore) in a dry blot system (GE healthcare). The blots were incubated with either mouse anti-ubiquitin P4D1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-HA 16B12, rat 3F10-POD conjugated, or M2 anti-Flag monoclonal antibodies (all from Merck, Darmstadt, Germany) and were processed as described [16 (link)].