Mouse anti his antibody

Escherichia coli DH10β cells carrying pJC78 were transformed with the construct pMLBADAtaC_1866–2428 [17 (link)], and cultured in LB broth with ampicillin 100 µg ml⁻¹, trimethoprim 20 µg ml⁻¹ at 37°C with shaking until an OD_{600 nm} of 0.4 was reached, followed by induction with 0.2% l-arabinose and 1 mM IPTG. After 16 h incubation, AtaC was purified. The bacterial cell pellet was isolated by centrifugation at 6000g for 10 min and lysed using a cell homogenizer (Stansted Fluidics Ltd. SPCH-10). Any intact cell debris was thereafter pelleted by centrifugation at 10 000g for 30 min before purification from the supernatant using an Ni–NTA (Qiagen, UK) gravity column (Thermo Scientific, USA).
Glycosylated product was analysed by SDS–PAGE and transferred onto a nitrocellulose membrane before being analysed by immunoblot using a mouse anti-His antibody (AbCam, UK) and an IRDye 680CW goat anti-mouse conjugate secondary antibody. Detection of fluorescent signal was carried out using a LI-COR imaging system.

SK-N-SH cells infected with RABV for 72 h were collected and lysed in NP-40 lysis buffer (Beyotime, Shanghai, China). Cell lysates were mixed with 4× loading buffer (Solarbio, Beijing, China) and boiled at 100 °C for 15 min. Protein samples and recombinant M protein samples were separated using SDS-PAGE with a 10% polyacrylamide gel [21 (link)] and transferred onto NC membranes (GE Healthcare, Amersham, UK). After blocking with 5% non-fat milk at 37 °C for 1 h, NC membranes were washed three times with PBST and incubated with the undiluted supernatant of hybridoma cells or mouse anti-His antibody (1:1000; Abcam, Cambridgeshire, UK) and anti-N mAb (produced in our laboratory) [22 (link)] as primary antibody at 4 °C overnight. Then, the membrane was incubated with HRP-conjugated goat anti-mouse antibody (1:10,000; KPL, Gaithersburg, MD, USA) as secondary antibody at 37 °C for 1 h. Membranes were then washed and the target protein bands were detected with enhanced chemiluminescence (ECL) (Vazyme, Nanjing, China).

Western blot analysis was used to confirm the expression of NIE recombinant protein. For this purpose, the method described by Sayadmanesh et al., was exploited (22 (link)). Briefly, following the electrophoresis of NIE protein on a SDS-PAGE, it was transferred on a nitrocellulose membrane. The membrane was blocked through an overnight incubation of the membrane in 5% w/v of Skimmed milk in PBST a 4 °C. Following the wash with PBST, the membrane was incubated in PBST containing mouse anti-His antibody (Abcam, USA, 1:5000). Then, the membrane was washed and incubated in PBST containing HRP-conjugated anti-mouse IgG and finally, the expression of the NIE protein was investigated by addition of substrate solution (DAB, Tris 50 mM, pH 8 and H₂O₂).