Mouse anti gfp antibody

Spores were fixed in 85% methanol and incubated with rabbit-anti-spore antibodies^{47 (link)}, diluted 1:10.000 in PBS with 5% bovine serum albumin (BSA) and with 1:2000 diluted Alexa488 conjugated goat-anti-rabbit-IgG (Thermo Fisher Scientific, Whaltham, MA). Cellulose was stained with 20 µg/ml Calcofluor White (Sigma-Aldrich, St. Louis, MO). For whole mount staining, structures developed on polytetrafluoroethylene membrane (Merck Millipore, Billerica, MA), were fixed with 50% and 100% methanol, successively, and stained with 1:2000 diluted mouse-anti-GFP antibody and 1:2000 diluted Alexa-Fluor594 conjugated anti-mouse antibody (Thermo Fisher Scientific, Whaltham, MA). Structures were mounted in the presence of 3 µM DAPI and imaged using a Leica LP2 confocal microscope.

Western blotting was done according to a standard method. The antibodies used were as follows: mouse anti-HA tag antibody (clone 6E2; Cell Signaling Technology [CST], Danvers, MA), mouse anti-Myc tag antibody (clone 9B11; CST), mouse anti-Flag tag antibody (clone M2; Sigma-Aldrich), rabbit anti-human phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit anti-human p44/42 MAPK (Erk1/2), rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-human SAPK/JNK (CST), rabbit anti-human phospho-Akt (Ser473), rabbit anti-human Akt, mouse anti-human C5aR antibody (Bio-Rad Laboratories, Hercules, CA), rabbit anti-human CXCR2 antibody (Thermo Fisher Scientific), rabbit anti-human CXCR6 antibody (GeneTex, Irvine, CA), rabbit anti-human CXCR7 antibody (Proteintech, Rosemon, IL), mouse anti-human CMTM6 antibody (used as ab1) (Absea Biotechnology, China), rabbit anti-human CMTM6 antibody (used as ab2) (Sigma-Aldrich), mouse anti-GFP antibody (Thermo Fisher Scientific), rabbit anti-GAPDH (CST), mouse anti-α-tubulin (Sigma-Aldrich), and mouse anti-β-actin (Sigma-Aldrich) antibodies.

FCU-GFP expression in transgenic parasite lines was verified by immunofluorescence imaging analysis. Mixed stage transgenic P. falciparum-infected erythrocytes were fixed with 4% formaldehyde/0.0075% glutaraldehyde (Polysciences, Inc.) using the method of Tonkin et al.[79 (link)]. Fixed cells were permeabilized with 0.01% Triton-X 100 (Sigma-Aldrich) and blocked with 10% normal goat serum and 3% BSA. To detect FCU-GFP localization, the suspension was incubated with a 1:1000 dilution of a mouse anti-GFP antibody (Thermo Fisher Scientific) followed by secondary antibody Alexaflour-488 rat anti-mouse (Molecular Probes) diluted to 1:500. Cells were resuspended in Slowfade Antifade reagent with DAPI to stain parasite nuclei (Thermo Fisher Scientific) and mounted on slides using Flouromount-GTM (Southern Biotech). Fluorescent microscopic images were obtained with an Olympus BX61 system and deconvoluted using SlideBook 5.0 software (Intelligent Imaging Innovations).

HCT116 WT and TBC1D15−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with Valinomycin for 3 hr and fixed for 30 min with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS. The fixed cells were washed four times with PBS, followed by permeabilization for 40 min with 0.1% Saponin and 5% goat serum in PBS. The cells were then incubated for 1 hr with mouse anti-GFP antibody (Invitrogen clone 3E6), followed by 1 hr with nanogold-conjugated anti-mouse IgG antibody (Nanoprobes) and further processing as described (Tanner et al., 1996 (link)). Thin sections (∼80 nm) were counter stained with uranyl acetate and lead citrate. The sections were examined with a JEOL 200 CX transmission electron microscope. Images were collected with a digital CCD camera (AMT XR-100; Danvers, MA).

Drosophila orthologs of human genes were identified via the comparative genomics section of the Ensembl project. TRiP RNAi lines were obtained from Bloomington Drosophila Stock Center. To knockdown genes in adult IPCs, TRiP RNAi lines were crossed to the UAS-Dcr-2.D; Ilp21 gd2HF(attP2) Ilp215–1-GAL4 strain. To knockdown genes in adult fat body, TRiP RNAi lines were crossed to the UAS-Dcr-2.D; Ilp21 gd2HF(attP2) Lk6-GAL4 strain. To measure circulating and total Ilp2HF content per fly, Ilp2HF ELISA^{10 (link)} was used with the following modifications: fifteen ad libitum fed 3-day-old male flies abdomens were dissected open, submerged in 50 μl of PBS with 0.2% Tween 20, and vortexed for 30 min at room temperature. The supernatants of hemolymph extraction were transferred to anti-FLAG antibody coated ELISA plate wells containing 50 μl of anti-HA-peroxidase at 2.5 ng/ml in PBS with Tween 20. Two CPTI protein trap lines for CG9650, CPTI000886 and CPTI001740, were obtained from Kyoto Stock Center. Immunostaining for GFP and Ilp2 protein in adult brains was performed as previously described^{10 (link)} with the following modifications: mouse anti-GFP antibody (1:1000; Invitrogen), rabbit anti-Dilp2 antibody (1:1000) (7), Alexa Fluor 488 and 647 secondary antibodies (1:1000; Invitrogen) were diluted and incubated in PBS with 0.3% Triton-X100.