Gt anti ms igg h l secondary antibody

Western blotting was performed to assess protein expression in the transgenic line, following the method optimized by Hao et al.^{42 (link)}, with slight modifications. Proteins of the OxF₃H line were collected at 2, 12, 24, and 36 h after WBPH infestation. Total protein was isolated with the 10 ml TCA/Acetone (10% Trichloroacetic acid (TCA); 0.07% β-ME in Acetone P.A.) method proposed by Xu et al.^{43 (link)}. Equal amounts of protein were boiled for 5 min and separated on 10% SDS–PAGE at 100v for 150 min, and then transferred to a NC membrane (Whatman Japan) by a semi-dry method running for 90 min at 19v, using a Trans-Blot DS semi-dry transfer cell (Bio Rad). The membrane was blotted in TBST (0.1% Tween 20 in TBS) and 5% non-fat dry milk (w/v) for 2 h at room temperature. Proteins were further blotted with primary rabbit anti-F3H antibodies in 5% non-fat dry milk (w/v) and TBST overnight at 4 °C, and rinsed three times for 10 min in TBST solution. The membranes were then incubated in Gt anti-Ms IgG (H + L) secondary antibodies (Invitrogen USA), at a dilution of 1:1,000, for 2 h at room temperature, and rinsed three times for 10 min in TBST solution. The blot was developed with Amersham ECL (GE Healthcare UK), and protein bands were exposed on X-ray film. Western blotting and quantification analyses were performed in at least two biological replications (Supplemental Figure S2).

The cell debris was prepared by homogenizing the induced cells, and the protein lysate was prepared using NP-40 buffer (150 mM NaOH, 1.0 % NP-40, 50 mM tris (pH 8.0)) containing protease and phosphatase. Their protein concentrations were quantitated using Quick Start Bradford 1X Dye reagent (Bio-rad). The samples were run on a 15 % SDS-PAGE, transferred to nitrocellulose membranes, and were then blocked with 3 % bovine serum albumin in T-TBS (20 mM Tris, 150 mM NaCl, 0.1 % Tween 20 detergent). The membranes were incubated overnight at 4 ℃ with Anti-HA tag (abcam, 1:500 dilution). The blots were washed and incubated for 1 h at room temperature with the Gt anti-Ms IgG (H+L) secondary antibodies (Invitrogen, 1:5000 dilution). Immunoreactive protein bands were visualized with ECL Prime Western Blotting Detection Reagents (Amersham) and light emission detected by exposing the membrane to X-ray film (AGFA).

HBP vacuoles were lysed, and the protein amount was estimated by Bradford protein assay. The samples were run on a 10% SDS–PAGE, transferred to nitrocellulose membranes and blocked with 5% skim milk in T–TBS. The membranes for HBP vacuoles were incubated overnight at 4 °C with Anti‐6X His tag antibody (abcam, UK, 1:1000 dilution). The blots were washed and incubated at room temperature for 1 h with Gt anti–Ms IgG (H + L) secondary antibodies (Invitrogen, 1: 5000 dilution). The membranes were washed and reacted with ECL STAR (DYNE, Korea). Immunofluorescent blots were developed by X‐ray film (AGFA).