Bradford reagent

To measure total protein content of adult female A. stephensi derived from control or ABA-treated larvae, 2 mosquitoes within 8 h of eclosion were homogenized in 10 mM dithiothreitol with 1 mM protease inhibitor cocktail (Sigma) in 100 μL loading buffer (Bio-Rad). Samples were boiled for 5 min and then centrifuged at 10,000 × g for 10 min at 4°C. Supernatants were transferred to new tubes and stored at −80°C. Total protein content was determined using Bradford reagent (Alfa Aesar) using bovine albumin serum (BSA) as a standard. Three separate cohorts of mosquitoes were used to complete biological replicates of these studies, each with three technical replicates.

After harvest, mouse tissues were snap-frozen in liquid nitrogen, and stored at −80 °C before lysis. Tissues were homogenized in a lysis buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 1 mM sodium ortho-vanadate, 10 mM sodium glycerophosphate, 50 mM sodium fluoride 5 mM sodium pyrophosphate, 0.27 M sucrose, 2 µM microcystin-LR, 1 mM benzamidine, 0.1% (v/v) 2-mercaptoethanol, 0.2 mM phenylmethanesulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin and 1 mg/ml aprotinin). Tissue homogenates were lysed on ice for 30 min, and tissue debris was removed through centrifuge to obtain tissue lysates. Measurements of protein concentrations were carried out using Bradford reagent (Thermo Fisher Scientific).

CD4⁺ T cells were lysated and proteins were extracted using a nuclear extraction reagent (Boster Biological Technology, Pleasanton, CA, USA). Proteins were quantified by the Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by 12% vertical dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred into a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, St. Louis, MO, USA). The PVDF membrane was blocked in 5% skim milk for 1 h at room temperature, then incubated with an antibody against P65 (GB11142, 1:1000, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) or P50 (ab7971, 1:5000, Abcam, Cambridge, MA, USA) for 12-16 h at 4℃ , and followed by incubating with a mouse anti-rabbit IgG antibody (H&L) (GenScript, Piscataway, NJ, USA). Proteins were detected with an enhanced chemiluminescence (ECL) western blot detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantification of P65 and P50 was normalized to GAPDH by densitometry.

Protein lysates were prepared in a RIPA lysis buffer using ice and a 27 G needle with the addition of protease (Roche, 11836170001, Indianapolis, IN, USA) and phosphatase (Roche 4906837001) inhibitors. Quantification was performed using a detergent-compatible Bradford reagent (Thermo Scientific, 1863028, Waltham, MA, USA). The proteins were loaded into a 12% stain-free gel, and blocking was performed using 3% low-fat milk for 2 h. The membrane was incubated overnight at +4 °C with the following antibodies: anti-PINK1 (Abcam, ab23707, Waltham, MA, USA) 1:1000, anti-LC3 (Cell Signalling, #3868) 1:1000, anti-FUNDC1 (Novus Biologicals, NBP1-81063, Centennial, CO, USA) 1:2500, and anti-p62 1:15,000 (Abcam, ab109012). The membranes were then washed 3 times in TBST and incubated with secondary antibodies (1: 200,000) for 1 h. Subsequent detections were accomplished using the Femto SuperSignal chemiluminescent reagent (Thermo Scientific, 34095). Because of the low protein yield on D1, Western blot was only performed on D7 and D14 of differentiation. The results obtained with the Western blot were normalized to the total protein amount. All images of the Western blots can be found in the supplementary data.

Poly (ethylene glycol) diacrylate (PEGDA, MW 575), poly (ethylene glycol) methacrylate (PEGMA, MW 360), 2-hydro-xy-2-methylpropiophenone (HMPP), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), mineral light oil, Span 80, Tween 20, BPA, glutaraldehyde (GA), fluorescein isothiocyanate (FITC) assay kit, and Pluronic F-127 were purchased from Sigma-Aldrich (St. Louis., MO, USA). Laccase (Lac, 1.07 U/mg) from Trametes versicolor was also purchased from Sigma-Aldrich and was used without further purification. Bradford reagent was purchased from Thermo fisher (Waltham, MA, USA). Other agents were at least of analytical grade. Citric phosphate buffer (50 mM) and ultrapure water were used in all experiments.

LAPC4 (1.0 x 10⁵ cells/well), LNCaP (1.0 x 10⁵ cells/well), and VCaP (1.6 x 10⁵ cells/well) were seeded in 24-well plates with media containing 5% charcoal stripped FBS and grown for 18 h before PSEBC-TSTA (3.5 MOI) adenovirus infection, DsiRNA transfection, and treatment with vehicle (EtOH) or DHT (10 nM). Seventy-two hours following infection, cells were washed once with HBSS and lysed. Luciferase assay was performed following manufacturer guidelines (Promega) with 20 uL of each lysate. Relative luminescence unit (RLU) was normalized by protein content in each well (normalized RLU = total RLU/total protein amount). Protein concentration was estimated by adding 250 μl of Bradford reagent (ThermoFisher Scientific) to 3 μl of total lysate. Absorbance was measured using an Infinite F50 absorbance microplate reader (Tecan, Mannedorf, Switzerland) at 595 nm.