Powerpac basic system

SMG-C6 cells were lysed in ice-cold RIPA buffer (GenDEPOT, Barker, TX; R4200-010) and protein concentrations were measured using s spectrophotometer (Nanodrop; Thermo Fischer Scientific, ND-1000). Protein samples were separated using 10% SDS-PAGE gels (Bio-Rad, Hercules, CA). After electrophoresis in a Power-Pac Basic system (Bio-Rad), proteins were transferred to nitrocellulose membranes using an iBLOT 2 Dry Blotting system (Thermo Fisher Scientific, IB21001). The membranes were blocked with 10% non-fat milk and incubated with anti-ERK antibodies (1:1000; Cell Signaling Technology, 9102) and anti-pERK antibodies (1:1000; Cell signaling, 9101) at 4 °C overnight. After washing, membranes were incubated with anti-rabbit IgG-HRP (1:5000; Santa Cruz Biotechnology, sc-2030). Immunoreactivity was visualized by ECL reagents (Thermo Fisher Scientific, 32106) and detected by the Chemidoc XRS+ system (Bio-Rad Laboratories).

Purified svCRiSPs (5 µg) were separated by a precast 4–12% (w/v) NuPAGE^® Novex Bis-Tris gels (Invitrogen™, Carlsbad, CA, USA) under non-reducing conditions. The gel was run at 100 V for 90 min with MES SDS running buffer using an XCell SureLock™ mini cell electrophoresis system (Invitrogen™) and a PowerPac Basic system (Bio-Rad Laboratories, CA, USA). SeeBlue^® Plus2 marker (Life Technologies™, Carlsbad, CA, USA) was used for estimated molecular weight. After electrophoresis, gel was visualized with SimplyBlue™ SafeStain (Life Technologies™).
Crotaline CRiSPs (4 µg) were subjected to SDS-PAGE and transferred to PVDF membrane (Millipore Immobilon, Carrigtwohill, Ireland) using a Transblot^® Semi-Dry Transfer Cell (Bio-Rad) at 100 mA for 90 min. The membrane was visualized with Coomassie brilliant blue R-250 staining (0.25% Coomassie Brilliant Blue R-250 in 40% methanol) for 5 min. Then, the protein bands were excised for N-terminal amino acid sequencing. The sequences of crotaline CRiSPs were determined using automated Edman degradation on a PPSQ-33B protein sequencer (SHIMADZU, Kyoto, Japan) according to the manufacturer’s instructions. The N-terminal amino acid sequences of svCRiSPs were identified and compared with other proteins using Basic Local Alignment Search Tool (BLAST—http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 April 2020).