Biochemical assays

Cell lysis

HEK 293T cells (90% confluent) or cerebellar granule neurons (5 x 10⁶ cells per well) were grown in 6-well plates for the desired time periods. After aspirating off the medium, cells were washed once with PBS and scraped in 100-300 µL of

2.2 Methods 35 lysis buffer (Table 2.2) supplemented with fresh protease inhibitors (1 mM DTT, 1 µg/mL pepstatin, 3 µg/mL aprotinin and 1 µg/mL leupeptin) on ice and further incubated for 30 min on ice. Following incubation in the lysis buffer, the samples were centrifuged (pre-cooled bench top centrifuge, Eppendorf, Germany) at 13,000 rpm for 10 min. The supernatant was collected in a fresh tube and protein concen-tration was determined using bradford assay (2.2.1).

Co-immunoprecipitation

Transfected HEK 293T cells were washed with PBS and lysed in coimmuno-precipitation (co-IP) buffer (Table 2.2) supplemented with fresh protease inhibitors (1 mM DTT, 1µg/mL pepstatin, 3µg/mL aprotinin and 1 µg/mL leupeptin). The cell lysates were incubated on ice for 30 min and centrifuged at 13,000 rpm for 10 min at 4^◦C. The supernatant was transferred to fresh tube and total protein amount was estimated using Bradford reagent. 1 mg of total protein lysate was incubated with 0.2-0.8 µg of antibody for 2 hrs at 4^◦C on rotor. 30 µg of total protein lysate was separately boiled with SDS-sample buffer and used as input. 20 µL of Protein A Sepharose beads (GE Healthcare), equilibrated with 1:1 co-IP buffer, was added to the cell lysate and incubated for 1 hr at 4^◦C on rotor. The lysate was then cen-trifuged at 13,000 rpm for 1 min and the pellet containing the beads was washed three times with lysis buffer. The final wash was done with PBS. The sample was spun down and the PBS was carefully removed. The protein bound to beads was eluted by boiling the samples in SDS-sample buffer at 95^◦C for 5 min. The samples were subjected to analysis by SDS-PAGE and western blotting.

Cell-based ubiquitination assay

Transfected HEK 293T cells were lysed in co-IP buffer supplemented with fresh protease inhibitors (1 mM DTT, 1 µg/mL pepstatin, 3 µg/mL aprotinin and 1µg/mL leupeptin) and 10 mM NEM. The cell lysates were incubated on ice for 30 min, centrifuged and the supernatant was transferred to a fresh tube. Total protein

2.2 Methods 36 concentration was estimated using the Bradford assay and 1 mg of total protein lysate was used for co-immunoprecipitation with the first antibody for 2 hrs at 4^◦C.

30 µg of lysate from each sample was boiled separately with SDS-sample buffer to serve as input. 20µL of Protein-A sepharose beads, equilibrated in co-IP buffer, was added to the lysates and incubated for 1 hr at 4^◦C. The samples were centrifuged at 13000 rpm at 4^◦C for 1 min and the pellet was washed with lysis buffer and PBS as described earlier. The beads were then boiled with SDS-sample buffer and the samples were analyzed with SDS-PAGE and western blotting by probing with anti-ubiquitin antibody.

26S proteasome inhibition

Cerebellar granule neurons were treated with vehicle (DMSO) or lactacystin (5µM) 10 hrs prior to lysis at DIV 3. Transfected cerebellar granule neurons (DIV 2) were treated with vehicle (DMSO) or lactacystin (5 µM) 10 hrs prior to lysis at DIV 6. The neurons were lysed in lysis buffer and equal amounts of protein were analyzed by SDS-PAGE and western blotting.

Luciferase assay

Expression plasmid encoding Renilla-Par6c and Renilla-Cyclin D1 fusion pro-teins were generated by Dr. Judith Stegm¨uller by cloning Renilla sequence upstream of Par6c and Cyclin D1, respectively, in pCMVmyc vector. HEK 293T cells were cultured on 24-well plate and transfected (3 wells/condition) with 2 µg of control vector or FBXO31 together with 0.1 µg Renilla-Par6c and 0.1 µg SV40 firefly lu-ciferase (pGL3 promotor) expression plasmid. The cells were lysed 2 days after transfection with 100 µL well of passive lysis buffer. The lysates were rocked for 20 min, centrifuged at 13000 rpm for 10 min and supernatant was transferred to fresh tube. 30µL of lysates per condition were loaded on a 96-well plate in triplicates and the plate was subjected to dual-luciferase assay with firefly and renilla substrates in the luminometer. Sabrina Galinski assisted in obtaining firefly and renilla luciferase

2.2 Methods 37 activity readings using WINGLOW software. The values were exported to Microsoft Excel for further analysis. The ratio of renilla to firefly activity indicated normalized protein expression of the respective constructs.

Purification of FBXO31 antigen

To generate the FBXO31 antigen, FBXO31 coding sequence (1224 -1521 bp en-coding 100 aa at C-terminus) was PCR amplified from wild-type BL/6 mouse cDNA and cloned into pET3a expression vector. E. coli BL21 cells were transformed with pET3a/FBXO31 expression plasmid and grown on 2xYT-agar plate with ampicillin.

Single colony was isolated, grown overnight in 50 mL 2xYT medium (supplemented with 50 µg/mL ampicillin) and appropriate volume was transferred to 1 L of fresh 2xYT to obtain an O.D. of 0.1. The culture was further incubated at 37^◦C to an O.D. of 0.6, induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown for additional 3 hrs. The cells were harvested by centrifugation and the pellet was lyzed in 20 mL BL21-lysis buffer with freshly added 1 mM PMSF by sonication. After centrifugation for 30 min at 37,000 rpm, the cleared lysate was incubated with 250 µL Ni-NTA Sepharose beads (Qiagen) and 1 mM imidazole and incubated overnight at 4^◦C on rotor. The sample was centrifuged at 4000 rpm and beads were incubated with 5 mL BC100 buffer with 20 mM imidazole for 30 min at 4^◦C on rotor. The sample was centrifuges again and 2.5 mL BC100 buffer with 40 mM imidazole was added to the beads. The beads were inbucated on rotor for 30 min at 4^◦C. In the last step, beads were washed in 250µL BC100 with 200 mM imidazole and the protein was eluted. The samples were subjected to analysis by SDS-PAGE as described previously.