Ecl plus detection reagent

Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer and boiled for 5 minutes. Protein was separated on either 10% or 8% SDS‐Page gels and transferred to PVDF Transfer Membrane Hybond^™ (Amersham Bioscience). Membranes were saturated with 5% non‐fat dry milk in PBS containing 0.1% Tween20 (PBST) for 1 hour at RT. The antibodies (anti ‐c‐MYC sc764, anti‐GFP sc9996, anti‐GST sc9996, anti‐E47 sc763, anti‐GAPDH sc‐32233 were from Santa Cruz Biotechnology; anti‐Flag F3165 was from Millipore; anti STRA8 Ab49405, anti‐SOHLH1 Ab41520 and anti‐HISTONE H3 Ab1791 were from Abcam; anti c‐KIT gently provided by Prof. S. Dolci) were diluted in PBST buffer and added to the PVDF membrane for 1 hour at RT or overnight at 4°C followed by incubation with the appropriate horseradish peroxidase‐conjugated secondary antibodies (Amersham Bioscience) for 45 minutes at RT. The STRA8 and SOHLH1 immunoprecipitated protein were detected after incubation with the peroxidase‐conjugated anti‐rabbit IgG light chain specific (Jackson ImmunoReasearch). All proteins were detected with ECL plus detection reagents (Amersham Bioscience) and visualized by chemiluminescence.

Proteins from CHIKV virions were detected using modified techniques previously described [29] (link), [30] (link). For reducing samples preparation, sample containing IMT and SGP11 virions were boiled for 5 minutes at 100°C in Laemmli buffer supplemented with 2% SDS and 1 mM DTT. Non-reducing samples were prepared in Laemmli buffer supplemented with 2% SDS but without DTT and were not boiled. SDS migration buffer was used for electrophoresis.
Proteins from CHIKV virions were separated by 10% SDS-PAGE respectively, and transferred to nitrocellulose membrane at 180 mA for 45 min in transfer buffer (24 mM Tris, 77 mM glycine, 20% methanol) using semi-dry transfer method. The membranes were blocked overnight in blocking buffer (TBST supplemented with 5% dry milk and 3% FBS), followed by 1 hour incubation at room temperature with either antigen-specific rabbit serum (1∶2000), or macaque serum samples diluted (1∶2,000) in blocking buffer. Antigen-specific sera against nsP1, nsP2, nsP3, nsP4, Capsid, E2 and E1 proteins were raised in rabbits by passive immunization. The appropriate HRP-conjugated anti-rabbit IgG or anti-monkey IgG secondary antibodies were then added and incubated for 1 hour, followed by chemiluminescence detection using ECL Plus detection reagents (Amersham Biosciences). Blots were exposed to films (Pierce, Thermo Scientific) and developed.

The samples were resuspended in sample buffer. The electrophoresis was done in 12% SDS PAG. After blotting, the nitrocellulose paper was put in BSA 3% overnight at 4 °C. The paper was put in 0.5 µg/mL biotinylated rabbit anti-GM-CSF antibody (Pharmingen, San Diego, CA, USA) for 2 h at 37 °C. After washing, the paper was incubated with 1:15,000 streptavidin conjugated peroxidase at room temperature for 1 h. The immunoblot was visualized using ECL Plus detection reagents (Amersham Pharmacia Biotech, Little Chalfont, UK) and developed on autoradiography film (Hyperfilm^TM ECL).