Sds nupage gradient gels

At the indicated time points medium was removed from BMM. Cells were lysed in 150 μl Nu PAGE LDS Sample Buffer (Life Technologies) by vigorous pipetting and scraping of the cells. Cell lysates were heated at 95°C and immediately placed on ice prior to loading onto 4–12% SDS NuPAGE gradient gels (Life Technologies). Western blots were generated from SDS-NuPAGE gels as previously described [11 (link)]. Blots were probed with antibodies to phospho-T180/Y182 p38, p38, phospho-T334 MK2 (27B7), MK2, and β-actin (13E5), (all from Cell Signaling Technologies). RNA was purified using RNeasy kits (Qiagen), cDNA generated using the Superscript VILO kit (Life Technologies) and real-time quantitative PCR was run using primer/probe sets (Table 1) (all from Life Technologies) and an ABI 7900HT (Life Technologies). Input RNA was normalized to GAPDH, and fold change of the indicated genes as compared to untreated, uninfected controls was quantified as ΔΔCT. The mRNA half-life (t_1/2) was calculated as −ln2/m, where m is the slope of the line fit on a semi-logarithmic plot of mRNA concentration as a function of time using least-squares regression as previously described [12 (link)].

At the indicated time points, medium was removed from BMDM. Cells were lysed in 150 µl 1X cell lysis buffer (Cell Signal Technologies, Danvers, MA)supplemented with PMSF (Sigma-Aldrich). Lysates were added to NuPAGE LDS Sample Buffer (Life Technologies), heated at 95°C for 10 min, homogenized by centrifugation using a QiaShredder (Qiagen, Valencia, CA) and immediately placed on ice prior to loading onto 4–12% SDS NuPAGE gradient gels (Life Technologies). Western blots were generated from SDS-NuPAGE gels as previously described (19 (link)). Blots were probed with antibodies to phospho-p44/42, p44/42, HIF-1α or β-actin (13E5), (all from Cell Signaling Technologies). RNA was purified using RNeasy kits (Qiagen), cDNA generated using the Superscript VILO kit (Life Technologies) and real-time quantitative PCR was run using primer/probe sets for pfkfb3 (ID number: Mm00504650_m1) (all from Life Technologies) and an ABI 7900HT (Life Technologies). Input RNA was normalized to HPRT. Fold change of the indicated genes as compared to untreated, uninfected controls was quantified as ΔΔCT.

phospho-Parkin wild-type or R104A (5 μM) buffer were incubated in ubiquitination buffer (30 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM ATP, 10 mM MgCl₂) with HsUBE1 (0.2 μM), UBE2L3 (2 μM) and Ub (20 μM). The reactions were quenched at indicated time points by addition of DTT- and iodoacetamide-containing LDS buffer and resolved on a 4-12% SDS NuPAGE gradient gels (Invitrogen) and transferred to a PVDF membrane (BioRad). Membranes were blocked in a 5% (w/v) milk solution in PBS-T (PBS + 0.1% (v/v) Tween-20) for 30 min and incubated overnight at 4 °C with a ubiquitin recognizing antibody (Ubi-1, NB300-130, Novus Biologicals) in 5% (w/v) BSA in PBS-T and 0.1% (w/v) sodium azide. The membrane was then washed with PBS-T, incubated for 1 h at room temperature with anti-mouse IgG-HRP (NXA931, GE Healthcare) in 5% (w/v) milk in PBS-T, washed in PBS-T and visualised using the Amersham Western Blotting Detection Reagent (GE Healthcare) and a ChemiDoc Touch Imaging System (BioRad).