Anti mouse igg nxa931

Virgins of the stock ${\displaystyle w[\ast ];;P\{w[+mC]=GAL4-elav.L\}}$ , $P\{w[+mC]=UAS-HsapSNCA.A53T\}$ were either crossed to males ${\displaystyle w[118];;P\{w[+]=UAS-AS69\}}$ , or $w[*];P(acman)]\{w[+]=UAS-GFP\}$ 5 (control). In the F1-progeny we selected for males with pan neural [A53T] $\alpha$ -synuclein and either AS69 or GFP concomitant expression. Climbing analysis was performed 5, 15 and 25 days post eclosion as previously described (Dinter et al., 2016 (link)). For each time point and per genotype 10 flies were analyzed in 10 tapping experiments with 60 s resting interval and the results averaged. The crosses were repeated n = 3 times.
In parallel, 10 fly heads from the F1-progeny and also from male w[*]; P(acman)w[+]=UAS GFP flies were homogenized in 100 μl RIPA buffer using the Speedmil P12 (Analytik Jena AG). The lysates were centrifuged at 12000 rpm for 10 min and the supernatant collected and used for immunoblot analysis. The following primary antibodies were used: mouse anti- $\alpha$ -synuclein (1:500, syn204, ab3309, Abcam) and mouse anti-syntaxin (1:500, 8C3, Developmental Studies Hybridoma Bank, Iowa, USA). Secondary antibody was anti-mouse IgG (NXA931) from GE Healthcare Life Sciences (1:5000).

Cells were harvested, centrifuged at 1000 rpm for 5 min, washed with phosphate-buffered saline (PBS), and cell pellets were resuspended in RIPA buffer (50 mM Tris-HCl Ph 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL® CA-630, 0.1% sodium dodecyl sulfate, 1 mM phenylmethanesulfonyl fluoride, cOmplete™ Protease Inhibitor Cocktail (all reagents supplied by Sigma-Aldrich). After 20 min incubation on ice, lysates were centrifuged at 13,000 rpm for 20 min to remove cell debris. Protein extracts were quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), according to manufacturer instructions. Protein extracts were loaded on 8% gels (SureCast™ Acrylamide Solution (40%), Invitrogen) and transferred to PVDF membranes (Fisher Scientific). Western blot analyses were performed using the following primary antibodies: c-MYB (sc-74512, Santa Cruz Biotechnology) and Actin (sc-1616, Santa Cruz Biotechnology). HRP-conjugated secondary antibodies used were anti-mouse IgG (NXA931, GE Healthcare, Fisher Scientific) and anti-rabbit IgG (NA934, GE Healthcare, Fisher Scientific). Antibody detection was performed with enhanced chemiluminescence (ECL) (Thermo Fisher Scientific).

Proteins of MV4-11, MOLM-13, and OCI-AML3 cells were extracted using the trichloroacetic acid method. Protein extracts were applied to a 4–20% SDS-PAGE (Thermo Fisher Scientific, Rockford, IL) followed by electrotransfer to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Blots were incubated with either rabbit anti-human GLI1 (C68H3, Cell Signaling Technology, RRID:AB_1903989) or mouse anti-human β-Actin (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189) at 4 °C overnight. The subsequent incubation with the peroxidase-conjugated secondary antibodies (anti-mouse IgG, NXA931, GE healthcare, RRID:AB_772209 and anti-rabbit IgG, 7074S, Cell Signaling Technology, RRID:AB_2099233) was followed by detection using ECL Western blotting detection reagents (GE Healthcare) and the FusionSL 4 3500 WL detection system (Vilber Lourmat, Sud Torcy, France).