Amersham protran premium 0.45 μm nc

To determine the expression and secretion of GFP fusion protein levels by recombinant A. oryzae in the larvae and the adult mosquito’s midguts, groups of 20–50 larvae and adult mosquitoes were examined at two time points (days 1 and 10) after inoculation at the larval stage and post emergence at the adult stage on days 1 and 10. Prior to dissection, individual larvae and adult mosquitoes were surface sterilized by washing in 75% ethanol and then rinsing in sterile PBS. The larvae and adult cohort that had been inoculated with A. oryzae wild type served as negative controls. Protein isolation from the dissected guts in larval and adult stages was performed according to the method of^{41 (link)}. Western blotting was performed as previously described^{42 (link)–44 (link)}. Protein samples were separated using 8% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Protran Premium 0.45 μm NC, GE Healthcare). The membrane was incubated in blocking buffer (0.1% Tween-20 with 5% w/v nonfat dried milk) and probed with GFP monoclonal antibody (GF28R), HRP (Invitrogen). Supernatant from cultures of A. oryzae producing GFP fusion protein was used as a positive control. Fungal cultures were centrifuged at 4000 × g for 15 min at 4 °C, and an equal volume of the culture supernatant was concentrated using Amicon Ultra-15 centrifugal filter units.

Total cellular proteins were extracted at 4 °C using erythrocytes lysis buffer (ELB) containing protease inhibitors (Roche, Basel, Switzerland). Proteins (10–20 μg) were resolved on 8–12% SDS–polyacrylamide gels and transferred to Amersham™ Protran™ Premium 0.45 μm NC (GE Health Care Europe, Freiburg, Germany).
Immunoblots were probed with antibodies against PD-L1 (E1L3N), JAK1 (6G4), phospho-JAK1 (Tyr1022/1023; D7N4Z), JAK2 (D2E12), phospo-JAK2 (Tyr1008; D4A8), phospho-STAT1 (Tyr701; 58D6; all by Cell Signaling Technology, Boston, MA, USA), STAT1 (10C4B40), phospho-STAT1 (S727; A15158B), IRF-1 (13H3A44; all by BioLegend, San Diego, CA, USA), monoclonal p53 (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA) or polyclonal p53 (#9282; Cell Signaling Technology). ß-tubulin (TUB2.1) or ß-actin (AC-15; both Sigma-Aldrich) served as loading control.

For western blot analysis, the cells were lysed in RIPA buffer (50 mM TRIS pH 7.5; 0.1% Triton X, 1 mM EDTA, 135 mM NaCl) supplemented with Protease Inhibitor Cocktail (cOmplete™ ULTRA Tablets, EDTA-free, glass vials Protease Inhibitor Cocktail, Roche, Switzerland). Three replicates of isolated proteins were pooled together and 20 μg of proteins in total was separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes (Amersham™Protran^® Premium 0.45μm NC; GE Healthcare, Chicago, IL, USA). The membranes were blocked for 1 h with TBS-T containing 5% milk (Milchpulver blotting grade, Roth, Karlsruhe, Germany) prior to incubation with primary antibodies. The primary antibodies used were diluted in blocking buffer as follows: 1:1000 anti-B4GALT1 (ab121362, Abcam, Cambridge, UK), anti-β actin 1:1000 (sc-69879, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Incubations were performed overnight at +4 °C. The membranes were then washed and incubated in HRP conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (ab205719 and ab6721, respectively, Abcam) for 1 h at room temperature. The signals were developed using Immobilon Western Chemiluminescent HRP Substrate (Merck, Germany) and photographed using the Alliance Q9 Advanced imaging system (Uvitec, Cambridge, UK). The protein signals were quantified using ImageJ [46 (link)].