Goat anti mouse secondary antibody

As mentioned above, at the end of co-culture period, K562 cells were collected, washed twice with cold PBS and lysed using RIPA-buffer for 30 min at 4°C. Homogenates were centrifuged at 13000×g for 15 min at 4°C and protein concentrations were calculated with the BCA protein Assay (Pierce, Rockford, IL, USA). Then, 50 μg of each protein sample was loaded on 12% SDS-PAGE and transferred to poly vinylidene difluoride (PVDF) membrane. The skim milk (5%) in TBS-T (20 mM Tris, 137 mM NaCl and 0.1% Tween 20) was used for blocking of the membranes for 60 min at 25°C. In the following, the membranes were incubated overnight at 4°C with primary polyclonal antibodies against β-actin (1:1000), BAX and Bcl-2 (1:500, Santa Cruz Biotechnology, CA), washed twice with TBS-T and were incubated with goat anti-mouse secondary antibody (1:5000 Santa Cruz) diluted in TBS-T for 60 min at 25°C. Next, the membranes were washed and protein bands were detected using enhanced chemiluminescence detection Kit (Roche, UK) with X-ray film. The intensity of protein bands was measured by ImageJ 1.6 software and signal intensity of each band was normalized to its corresponding β-actin control [26 (link)].

DNA constructs and vectors were used as described before [9] (link), [25] (link). FLT3-I867S and FLT3-D839G constructs were generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). Denotation: W78: ITD1, Npos: ITD2, W51: ITD3. Figure S1 displays the locations and insertions respectively substitutions of the analyzed mutations.
The following antibodies were used: FLT3 (S18) and goat anti-mouse secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA). AKT, MAPK, pSTAT5 (Tyr694), pAKT (Ser473) and pMAPK (Thr202/Tyr204) all from Cell Signaling Technology (Danvers, MA). STAT5 from R&D Systems (Minneapolis, MN). β-actin and goat anti-rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO). CD-135-PE from Beckman Coulter (Brea, CA). IgG1 PE Isotype control from BD Pharmingen (BD Bioscience, Franklin Lakes, NJ).

To investigate the Wnt-5a/β-catenin and P53 signaling pathways involved in BMSCs effect on K562 cells, these protein expression was assessed by western blotting. For this stage, K562 cells protein in both groups (control and experimental) were extracted and 50 μg of each cell protein sample was electrophoresed on 12% polyacrylamide slab gels and transferred to poly vinylidene difluoride membrane. In the following, the membrane was incubated with primary antibodies of Wnt-5a, β-catenin and P53 (1:500, Santa Cruz Biotechnology, CA) and was incubated with goat anti-mouse secon­dary antibody (1:5000 Santa Cruz) for 60 minutes at 25°C. Also, β-actin was used as the internal control to normalize. Next, the protein bands detected with X-ray film. Protein bands intensity were measured and then calculated the ratio of target protein/β-actin and the obtained values were graphed.

Total protein from cultured bGCs was isolated using 1× PLB (Promega Corporation, USA). The total protein concentration was determined using the Bradford method [39 (link)]. Western blotting was performed as described previously [12 (link), 13 (link)]. The antibodies used were (Santa Cruz Biotechnology Inc., Germany): anti-ACTR-II2A goat polyclonal antibody (product no. sc-5667), anti-SMAD7 rabbit polyclonal antibody (product no. sc-11,392), anti-PCNA rabbit polyclonal antibody (product no. sc-7907), anti-STAR rabbit polyclonal antibody (product no. sc-25,806), anti-SMAD2/3 rabbit poly clonal antibody (product no.sct-5678), anti-psmad2/3 rabbit monoclonal antibody (sct-8828) or anti-β-ACTIN mouse monoclonal antibody (product no. sc-47,778). At the end of the incubation period, the membrane was washed six times with 1× TBST and incubated with the corresponding donkey anti-goat, goat anti-rabbit, or goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The detection of the protein signal was then performed using Clarity Western ECL Substrate (Bio-Rad Laboratories). The relative band intensity was determined by ImageJ program (https://imagej.nih.gov/ij/).

After treatment shown above was over, cells were washed with PBS three times and were lysed with RIPA lysis buffer supplemented with the protease inhibitor (PMSF) and incubated on ice for 30 minutes. Supernatant was collected after centrifugation (12,000 rpm, 15 min), and protein concentration was determined by using the BCA protein assay kit according to the manufacturer's recommendations. The lysate was boiled with loading buffer and separated with 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with blocking buffer (5% skim milk powder in TBST (0.1% Tween 20 in Tris-buffered saline (TBST)) for 1 h, primary antibody 6 × HisTag (mouse polyclonal, Cell Signaling Technology, Boston, MA; 1:1000) was incubated overnight at 4 °C. After washing PVDF membrane three times with TBST, goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Dallas, TX; 1:1000) coupled with horseradish peroxidase (HRP) was incubated for another 1 h at room temperature. Anti-β-actin-HRP (Santa Cruz Biotechnology, Dallas, TX; 1:1000) was used as a loading control. Chemical reaction light signal detection was performed using the Clinx ChemiScope 3000 mini enhanced chemiluminescence (ECL) detection reagent.