Monoclonal anti flag m2 peroxidase antibody

Western blot signals of FLAG, tubulin, and SmD2 were detected using Monoclonal anti-FLAG M2-Peroxidase antibody (Sigma), mAb DM1A (Sigma) and anti-SNRPD2 antibody (Abcam), respectively. Northern analyses were performed by transferring RNAs from 8 M Urea-PAGE gel to Hybond-N membrane (Amersham) and probed by ³²P-labeled antisense DNA oligonucleotides that are listed in Supplementary Data 5.

The expression of plasmid-encoded sphingolipid kinases was determined using the protocol of Zhang et al. [12 (link)]. Yeast cells expressing deletion mutants of N-terminal FLAG-tagged human SphK2 were grown overnight on SC-URA media with 2% glucose. The cultures were diluted 1:100 into SC-URA media supplemented with 2% galactose in presence of an SphK2 inhibitor (0.5 μM SLM6031434) for 24–48 hours. Cells from 1 mL culture fluid were collected by centrifugation, re-suspended in 2 mL of 2M lithium acetate and incubated on ice for 5 minutes. This procedure was repeated and cells were suspended in 2 mL of 0.4M NaOH and incubated on ice for 5 minutes, re-centrifuged and the pellets were re-suspended in 0.25 mL of 1X Laemmli buffer and heated to 95°C for 5 minutes. After clarification by centrifugation, the supernatant fluid (10–15 μL) was loaded on 4–20% polyacrylamide gels and the resolved proteins were transferred onto a nitrocellulose membrane. Membranes were blocked with 5% (w/v) non-fat dried skimmed milk powder in TBS (Tris-buffered saline, pH 7.4) containing 0.1% Tween 20 for 1 hour at room temperature. After rinsing, membranes were incubated with monoclonal anti-FLAG M2-peroxidase antibody (Sigma-Aldrich #A8592) for an additional hour. After washing three times in TBS, the blot was developed by chemiluminesence using a commercial kit (PerkinElmer Western Lightning).