Calmodulin

HEK293 cells (4× P100), grown in Dulbecco's Modified Eagle Medium (DMEM), transfected with the plasmids expressing p85-wt-TAP or p85-A951E-TAP proteins, were harvested 24 h post-transfection. The complexes associated to the TAP-tagged constructs were purified as described (33 (link)). Briefly, the supernatant of the first IgG Sepharose purification was subsequently subjected to a second Calmodulin (Agilent Technologies) purification step. Purified proteins were precipitated with 10% trichloroacetic acid at 4°C overnight, pelleted at 14 000 g for 15 min at 4°C, washed three times with 1 ml of acetone and dissolved in SDS-loading buffer. An aliquot (25%) was analyzed on silver stained SDS-PAGE gels to visualize the purification of proteins associated to Gemin5 p85-wt-TAP or p85-A951E-TAP polypeptides. Immunodetection of Gemin5 and p85 was performed using anti-Gemin5 (Novus) antibody.

HEK293 cells were cultured with DMEM supplemented with 5 % fetal calf serum. Monolayers grown at 80 % of confluency were transfected with the constructs G5_845-1508-TAP and were harvested 24 h post-treatment (hpt). The complexes associated with the TAP-tagged proteins were purified as described [11] (link). Briefly, the extract from the TEV protease digestion of the first IgG Sepharose (Cytiva) purification was subsequently subjected to a second calmodulin (Agilent Technologies) purification step. Purified proteins were precipitated with 10 % trichloroacetic acid, washed with acetone, and dissolved in SDS–loading buffer. An aliquot (20 %) was analyzed on silver-stained SDS–polyacrylamide (PAGE) gels to visualize the purification of proteins associated with G5_845-1508-TAP polypeptides.

HEK293 cells (4 × P100 plates), grown in Dulbecco's Modified Eagle Medium (DMEM), transfected with the TAP-Gemin5 plasmids, were harvested 24 h post-transfection. The protein complexes associated to TAP-Gemin5 were purified as described (40 (link),41 (link)). An optional treatment with RNase A (75 μg/1.5 ml) 30 min at room temperature immediately following TEV protease (Life Technologies) digestion (41 (link)) eliminates the factors bound by RNA bridges; this concentration of RNase degrades all RNA probe in UV-crosslink assays. The supernatant of the first purification, treated or untreated with RNase A, was subsequently subjected to a second Calmodulin (Agilent Technologies) purification step. Purified proteins were precipitated with 10% trichloroacetic acid at 4°C overnight, pelleted at 14 000 g 15 min at 4°C, washed three times with 1 ml of acetone and, finally, dissolved in SDS-loading buffer. A small aliquot was analyzed on silver stained SDS-PAGE gels to visualize the purification of proteins associated to TAP-Gemin5.