Nupage lds loading buffer

Cultures of late-stage parasites (35–45 hpi) were first treated with 0.05% saponin and washed twice with PBS to enrich for parasites. The pellet was then resuspended in 1 ml of 0.5% Triton X and spun down hard. The supernatant was discarded and the pellet was resuspended in 2% SDS before being spun down hard again. The SDS extract was then used for SDS-PAGE while the pellet was discarded. The SDS extracts were mixed with 4× NuPAGE LDS loading buffer (Invitrogen) and NuPAGE sample reducing agent (Invitrogen) in 1:15 v/v ratio, heated at 100 °C for 10 min, cooled and loaded into the wells of the pre-cast NuPAGE Novex 4–12% Bid-Tris gels (Invitrogen). NuPAGE MOPS running buffer (Invitrogen) was used, as well as pre-stain plus (Fermentas) protein ladder.

HEK-293T cells were seeded in 10-cm dishes and transfected with FLAG-LRRK2 in the presence of myc-TRIM1 or myc-alone vector control. After transfection for 8 h, cells were treated with 100 nM bortezomib for 12 h. 20 h after transfection, cells were lysed as above, and lysate protein concentrations were normalized using BCA assay. FLAG-LRRK2 immunoprecipitation was performed using 30 μl FLAG antibody-conjugated beads (Sigma-Aldrich) at 4°C for 1 h. FLAG-LRRK2 was eluted by incubation with 100 µg/ml 3× FLAG peptide at 4°C for 1 h. 50 μl TUBE2 magnetic beads (LifeSensors) was washed 3× in TBS-T, added to eluted FLAG-LRRK2, and rotated at 4°C overnight. TUBE2 beads were washed 3× (50 mM Tris, pH 7.5, 250 mM NaCl, 0.2% NP-40, and 1 mM DTT), and bound proteins were eluted by heating at 70°C for 10 min in 20 μl 4× Nupage LDS loading buffer (NP0007; Invitrogen) containing 5% β-mercaptoethanol. Standards used were K48- and K63-linked recombinant polyubiquitin chains (R&D Biosystems).

Primary NSCs were lysed in prechilled RIPA lysis buffer (500 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Na deoxycholate, 1% NP40, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Sigma cat# 11697498001) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific cat# 78420). Cell lysates were incubated for 15 minutes on ice and cleared using 10-minute 15,000 rpm centrifugation at 4C. Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific cat# PI23225). Protein lysates were mixed with 4× NuPage LDS loading buffer (Invitrogen), heat denatured, loaded on a 4% to 12% SDS polyacrylamide gradient gel (BioRad cat # 3450124), and subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk in TBST and incubated with primary antibody at 4°C for 16 hours. Horseradish peroxidase–conjugated secondary antibodies and an ECL kit (BioRad cat # 1705060 and 1705062) were used to detect protein signals. Multiple exposures were taken on a BioRad ChemiDoc. Images were exported (300 dpi) and quantified using the Gels tool in FIJI/ImageJ (Version 2.1.0). GAPDH, total Erk, and Akt bands were used for normalization.

For glycosylation analysis, N-linked oligosaccharides removal was carried out using recombinant PNGase F and Endo H (New England BioLabs, Beverly, Massachusetts) according to the manufacturer’s instruction. Briefly, 2.25 μg of purified swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/1/2005 (H5N1) rNAs were combined with 1 μl of 10X Glycoprotein Denaturing Buffer and then denaturated by heating reaction at 95°C for 5 min. Proteins were chilled on ice and centrifuged for 10 sec before adding 2 μl of 10X G7 Reaction Buffer, 2 μl 10% NP40, 6 μl H₂O and 1 μl PNGase F to achieve a final volume of reaction of 20 μl. Samples were gently mixed and incubated at 37°C for 1 h. To assess the extent of deglycosylation, the proteins were mixed with 1X NuPage LDS loading buffer and 1X NuPage Sample reducing agent (Life Technologies), heated at 100°C for 5 min and finally loaded onto a 4–12% NuPAGE Bis-Tris gel (Life Technologies) subjected to SDS-PAGE. The mobility shifts were checked by Comassie staining.