Rabbit anti phospho s6k thr398

We used our previously generated antibody against Drosophila Mkrn1 [37 (link)]. Protein extracts from the head, thorax, abdomen, and ovaries of adult female Drosophila were prepared using lysis buffer (10 mM HEPES [pH 7.5]; 50 mM KCl; 10% glycerol; 5 mM Tris-HCl [pH 7.5]) with freshly added 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, protease inhibitor (Sigma), 1 mM Na₃VO₄, and 0.25 mM NaF (final concentration). For p-AKT and p-S6K analysis, phosphatase inhibitor cocktail 2 and 3 (Sigma) were used instead of Na₃VO₄ and NaF. The protein extracts were resolved by SDS-PAGE and the resulting blots were probed using the following primary antibodies: rabbit anti-Mkrn1, 1:3000 [37 (link)]; rabbit anti-phospho-AKT (Ser505), 1:1000 (Cell Signaling Technology, 4054); rabbit anti-AKT, 1:1000 (Cell signaling Technology, 9272); rabbit anti-phospho-S6K (Thr398), 1:1000 (Cell Signaling Technology, 9209); guinea pig anti-dS6K, 1:3000 [38 (link)]; mouse anti-Armadillo, 1:1000 (DSHB, N2 7A1); rabbit anti-p44/42 MAPK (Erk1/2), 1:2000 (Cell Signaling Technology, 9102). Band intensities were quantified using ImageJ software.

Fat bodies of mosquitoes were dissected 12 h or 24 h post blood meal. Proteins of 10 mosquito fat bodies were extracted in 100 μl lysis buffer (50 mM Tris, pH 7.4; 1% IGEPAL 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1× protease inhibitor mixture; 1× phosphatase inhibitor mixture) [9 (link)]. Immunoblotting was performed using standard procedures. Antibodies used for TOR signaling were rabbit anti-phospho-S6K (Thr398) (1:1000) (Cell Signaling), rabbit anti-S6K (1:1000), and rabbit anti-actin (1:2000) (Sungenebiotech, China). Protein used for immunoblotting for p-Akt was extracted from fat bodies/ovaries, and midgut 12 hpi. The p-Akt was detected using a Phospho-Akt (Ser473) Antibody (1:200) (Cell Signaling) [51 (link)]. Immunoblotting for TEP1 was performed similarly, except that proteins from ten whole mosquitoes were extracted in cracking buffer (8 M urea, 2% SDS, 5% β-mercaptoethanol, 125 mM Tris-HCl) and 1:1000 anti-TEP1 rabbit polyclonal antibody was used. Intensity of the signals was quantified by ImageJ software [52 (link)].