Polyvinylidene fluoride transfer membrane

Adult flies were decapitated with a razor blade. Heads were homogenized on ice using a motorized pellet pestle in lysis buffer containing 50 mM Tris-HCl pH 6.8, 130 mM NaCl, 1% Triton, 1 mM MgCl, and protease inhibitor complete (Roche). After a 30-min extraction on ice, the homogenate was cleared by centrifugation for 20 min at 3000 × g. Supernatant was resuspended in LDS sample buffer (Invitrogen) and denatured for 10 min at 70°. A volume of supernatant that corresponds to 15 heads was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 3–8% NuPAGE Tris-Acetate gels (Invitrogen) and blotted onto polyvinylidene fluoride transfer membrane (Millipore). Protein bands were visualized with a Ponceau S stain (0.1% Ponceau S and 0.5% acetic acid). Blots were blocked in TBST+5% milk and the membrane was incubated overnight with mouse anti-HA (Clone 16B12, Covance) diluted 1:500 in TBST+1% BSA or with antisynapsin [3C11 (anti-SYNORF1), Developmental Studies Hybridoma Bank, Iowa, City, IA] diluted 1:500 in TBST+1% BSA. Peroxidase-conjugated secondary antibodies and ECL plus system (Pierce) were used for detection.

For the Western blot assay, 3 × 10⁵ Saos‐2 cells per well were cultured in 6‐well plates, 24 hours before the transfection. We transfected 2 μg of the DKK1 expression plasmid (WT or the missense variant: p.Ala41Thr, p.Tyr74Phe, p.Pro84Leu, p.Ala106Thr, p.Arg120Leu, p.Ser157Ile). For the negative control, we transfected 2 μg of the empty pcDNA3 vector. Fugene HD was used following the manufacturer's instructions. After 24 hours, the medium was changed, reducing from 2 to 1 mL of DMEM, without FBS or antibiotics. Forty‐eight hours after transfection, the supernatant (conditioned medium) of each condition was collected and were concentrated using 10 K Amicon Ultra filters (Millipore, Watford, UK). Extracellular proteins (concentrated conditioned medium) were quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins from concentrated conditioned medium (5 μg/lane) were separated by electrophoresis in an SDS‐PAGE and transferred to a hydrophobic polyvinylidene fluoride transfer membrane (Millipore). The ab109416 antibody against DKK1 (Abcam, Cambridge, UK) was used. The images were developed using a peroxidase‐conjugated secondary antibody (anti‐rabbit IgG; A0545) for DKK1 antibody. All the experiments were performed in three independent biological replicates.

To evaluate the activation status of the mutant c-KIT, each cell line was seeded in 6-well plates at 5.0 × 10⁵ cells/well and incubated for 3 h in the plain RPMI. After incubation, cells were stimulated with fSCF (100 ng/ml) for 5 min and harvested and lysed with NP40 lysis buffer [1% NP 40, 10 mM Tris HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan), 1 mM Na₃VO₄, and 50 mM NaF]. The resulting protein lysates were loaded on SDS–polyacrylamide gels and electrophoresed followed by blotting to polyvinylidene fluoride transfer membrane (Merck, Darmstadt, Germany). Then, the membrane was blocked with 5% skim milk blocking buffer, followed by incubation with the primary antibody, rabbit polyclonal anti-phospho-c-KIT antibody (Tyr719) (Cell Signaling Technology, Danvers, MA, USA), goat polyclonal anti-c-KIT antibody (M-14, Santa Cruz Biotechnology), or mouse monoclonal anti-beta actin antibody (Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated antibodies, including donkey anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), goat anti-mouse IgG-HRP (Bio-Rad Laboratories) and goat anti-mouse IgG-HRP (Bio-Rad Laboratories) were used as secondary antibodies. The specific proteins were detected using the ECL reagent (PerkinElmer, Waltham, MA, USA).

For western blot analyses, whole cell lysates and the immunoprecipitates were subjected to SDS-PAGE and then transferred onto Polyvinylidene Fluoride transfer membrane (Merck-Millipore, Darmstadt, Germany). The membranes were immunoblotted with specific antibodies as indicated and then incubated with horseradish peroxidase-conjugated antibody against mouse or rabbit immunoglobulin (GE Healthcare), followed by detection with Immobilon Western (Merck-Millipore).
The following were used as primary antibodies in this study: anti-Flag M2 monoclonal (F3165, Sigma-Merck-Millipore), anti-T7-tag monoclonal (69522, Merck-Millipore), anti-β-actin (A2066, Sigma-Aldrich), and anti-cyclin B1 (sc-245, Santa Cruz Biotechnology). Secondary antibodies: the horseradish peroxidase (HRP)-conjugated anti-mouse IgG (NA931V, GE Healthcare), HRP-conjugated anti-rabbit IgG (NA934V, GE Healthcare). We tried to assess the reported specificity of anti-ZFP36L2 antibodies, but failed to detect reliable cell endogenous antigen signals with the commercially available antibodies.

Cells were rinsed twice with ice-cold PBS and lysed in Giordano buffer (50 mM Tris-HCl pH7.4, 250 mM NaCl, 0.1% Triton X-100 and 5 mM EDTA; supplemented with phosphatase- and protease inhibitors). Equal protein amounts were separated using SDS-PAGE and blotted onto polyvinylidene fluoride transfer membranes (Millipore, Darmstadt, Germany). After blocking the membranes in TBST (10 mM Tris-HCl pH8.0, 150 mM NaCl, 0.2% Tween 20) containing 10% milk, membranes were incubated with the proper primary antibodies (listed in Table 2) and appropriate HRP-conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA). Bands were visualized using chemoluminescence and visualized by exposure to X-ray film.