Gammabind plus sepharose beads

The association between recombinant hnRNP R and SMN was analyzed by coimmunoprecipitation using GammaBind Plus Sepharose beads (GE Healthcare). 250 or 500 ng of rhnRNP R and 250 ng of rSMN were incubated in binding buffer, comprising 50 mM sodium phosphate (pH 8.0), 5% (v/v) glycerol, 50 mM NaCl and 0.1% Tween, with 20 µl Sepharose beads and 1 µg antibodies against hnRNP R (ab30930, Abcam), SMN (610647, BD Bioscience) or non-specific IgG control (anti-GFP, sc-8334, Santa Cruz) for 1 h at RT. The resin was washed 5 times with binding buffer to remove unbound proteins. For elution beads were boiled in 2xLaemmli buffer at 95°C for 5 min. The eluted proteins were then analyzed by Western blotting (for more information see the chapter Western blotting). Notably, Light chain-specific secondary antibodies (Jackson Immunoresearch) were used for detection since the 55 kDa heavy chain from the immunoprecipitation would mask the SMN signal.

Alvocidib/flavopiridol (S2679) and AT7519 (S1524) were from Selleckchem (Houston, TX, USA). Glutathione Sepharose 4B (17-0756-01) and Gammabind Plus Sepharose beads (17-0886-01) were from GE Healthcare (Chicago, IL, USA). Anti-FLAG tag affinity beads (ab270704) were from Abcam (Cambridge, MA, USA). pEBG-GST-CILK1/ICK encoding GST-CILK1/ICK was described in [13 (link),21 (link)]. pCMV6-Myc-Flag-CILK1 encoding CILK1-Myc-Flag (RC213609) was from Origene (Rockville, MD, USA). pCIG-HA-KIF3A encoding HA-KIF3A was described in [35 (link)].

All ELISAs were performed using Costar half-area, high-binding plates. For worm-specific IgE, plates were coated with 20 μg/mL LsAg in PBS and incubated overnight at 4°C. Plates were blocked with PBS/5% BSA and 0.05% Tween 20. IgG was depleted out of plasma using GammaBind plus Sepharose beads (GE Healthcare Biosciences). Plasma was then diluted in 1% BSA/PBS and 5-fold serial dilutions were made. Samples were added to the plate in duplicate. Plates were then washed and plate-bound IgE detected by the addition of biotinylated anti-mouse IgE (BD Biosciences) diluted in 1%BSA/PBS. Following another wash, alkaline phosphatase conjugated streptavidin (Jackson Immuno Research Labs) diluted 1:1000 in 1%BSA/PBS was added. Plates were developed by the addition of 4-nitrophenyl phosphate disodium (4-NPP, Sigma-Alderich) in 0.1M carbonate buffer. Absorbance was read at 405nm using a Victor³ V microplate reader from PerkinElmer. Detection of total IgE was performed with identical steps as for parasite-specific IgE except that plates were initially coated with 10 μg/mL anti-IgE (BD Biosciences)in PBS and plasma samples were plated at dilutions of 1:10 and 1:90. Total IgE concentrations were determined using an IgE standard curve (BD Biosciences) and WorkOut 2.0 ELISA software (PerkinElmer).

Purified Ozz–EloBC and Cul5–Rbx1 were mixed at a 1:1 ratio and incubated on ice for 1 h. The mixture of the two subcomplexes was resuspended in IP buffer (50 mM Tris HCl pH 7.6, 150 mM NaCl, 500 mM EDTA and 01% NP-40). Gammabind Plus Sepharose beads (GE Healthcare) were washed three times with IP buffer and added to the Ozz ligase and incubated for 1 h. Preclear Ozz ligase was incubated with 2.5 μg of anti Elongin C (BD Bioscience) and 5 μg of Rbx1 (Neomarkers) antibodies for 2 h at room temperature (RT). Samples were immunoprecipitated with Gammabind Plus Sepharose (GE Healthcare) for 1 h a RT. The beads were washed three times with IP buffer and once with IP buffer without detergents. Bound proteins were released by boiling the beads with sample buffer and separated on SDS–polyacrylamide gels under denaturing conditions, followed by SYPRO Ruby Protein Gel Staining (ThermoFisher Scientific).

Thyroid cells were plated in T75 flasks and allowed to grow until 70% confluence. Complete media was replaced with 5% charcoal dextran-treated FBS-containing media and flasks were incubated for 24 hours. Cells were treated for an additional 24 hours with +/− 10 μM VMR. Cells were collected by gentle scraping, spun down, and subjected to 1% NP-40 lysis buffer on ice for 10 minutes with periodic mixing. Samples were centrifuged for 10 minutes at 14,000 rpm and 4°C and supernatants were collected. GammaBind Plus Sepharose beads (GE Healthcare) were washed 3 times with PBS. CRAF antibody (BD Bioscience) was added to beads and the mixture was incubated for 2 hours at 4°C with constant rotation. At the same time, 100 μg of sample lysate was also incubated for 2 hours at 4°C with constant rotation to pre-clear lysates. The beads coated with antibody were washed three times with PBS before the pre-cleared lysate was added to them. This mixture was incubated overnight in eppendorf tubes at 4°C with constant rotation. The tubes were centrifuged for 10 minutes at 14,000 rpm and beads were washed three times in 1% NP-40 lysis buffer. Loading buffer was added to the samples. Samples were boiled for ten minutes and spun down. Supernatants were subjected to 12% SDS-polyacrylamide gel electrophoresis as in our prior studies and probed for CRAF with primary antibody (Cell Signaling).

Transgenic flies expressing BRM fused to FLAG epitope tags were entrained in 12 hr light:12 hr dark (LD) conditions at 25°C for three days and collected on the fourth day. Fly head collection and protein extraction with RBS buffer with sonication were performed as described above. Extracts were quantified and equal concentrations were subjected for IP. Samples were pre-cleared using sepharose beads (Sigma) to reduce nonspecific binding. Co-IPs were performed as described for S2 cell experiments except α-FLAG M2 (Sigma), α-PER (GP5620), α-TIM (R3), and α-CLK (Santa Cruz Biotechnology H3107) antibodies were used. Samples were incubated with antibodies for 4 to 6 hours at 4°C on an end-over-end rotator. 20 μl of GammaBind Plus sepharose beads (GE) was added and incubation was continued for 2 hours. Samples were washed with RBS buffer three times, 10 minutes each, and immune complexes were resolved by SDS-PAGE as described above.