Mouse anti c myc sc 40

For immunofluorescence, nephrocytes were dissected, fixed for 20 min in PBS containing 4% paraformaldehyde, and stained according to the standard procedure. The following primary antibodies were used: rabbit anti-sns (Bour et al., 2000 (link)) (1:300, gift from S Abmayr) and guinea pig anti-Kirre (Galletta et al., 2004 (link)) (1:200, gift from S Abmayr). Other antibodies used were rabbit anti-Rab5 (ab18211, abcam, 1:100), mouse anti-Rab7 (Rab7, DSHB 1:100), mouse anti-Myc (9E10; DSHB, 1:100), mouse anti-c-Myc (sc-40; Santa Cruz Biotechnology 1:100), and rabbit anti-RAB11 (#5589S; Cell Signaling Technology, 1:100). The following secondary antibodies were used: Alexa Fluor 488 donkey anti-rabbit (#A-21206, Thermofisher, 1:200), Alexa Fluor 488 donkey anti-mouse (#A32766, Thermofisher, 1:200), Alexa Fluor 568 donkey anti-rabbit (#A10042, Thermofisher, 1:200), Alexa Fluor 568 donkey anti-mouse (#A10037, Thermofisher, 1:200), Alexa Fluor 568 goat anti-guinea pig (#11075, Thermofisher, 1:200).
Apoptotic cells were visualized using the In Situ Cell Death Detection Kit (#11684795910, Sigma/Roche) according to the manufacturer’s instructions. For imaging, a Zeiss LSM 880 laser scanning microscope was used. Image processing was done by ImageJ and GIMP software.

Cell pellets were collected by centrifugation and snap‐frozen on dry ice. Cells were thawed on ice and lysed by shaking with acid‐washed glass beads in a FastPrep FP120 device (Savant) at speed 5 for 30 s. Lysis was performed in lysis buffer A (50 mM NaCl, 50 mM Tris pH 7.6, 0.2% Triton X‐100, 0.25% NP40) containing phosphatase inhibitor cocktail 04906837001 and protease inhibitor cocktail 04693159001 (Roche). Protein concentration was determined using the BCA assay. Equal protein concentrations of each sample were loaded and proteins were separated on a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and blotted onto nitrocellulose membranes.
HA epitope‐tagged versions of Aph1, Rad1, Hus1‐, and Rad9 as well as Chk1 were detected by mouse anti‐HA Sc7392 (Santa Cruz Biotechnology) or mouse anti‐HA 2367 S from Cell Signaling Technology (Bionordika AB, Stockholm, Sweden). Cds1 was detected using Mouse anti‐c‐myc Sc40 (Santa Cruz Biotechnology). To be able to detect the low‐abundance Aph1 protein, incubation with the primary antibody was for 72 h. The loading control was α‐tubulin detected by mouse anti‐α‐tubulin T5168 (Sigma), or staining of total protein with Ponceau S Solution (Sigma). The secondary antibody was horseradish peroxidase‐coupled α‐mouse A4416 (Sigma).

CD44+CD24− MCF-7/CSCs and SKBR3/CSCs were transfected with, or without, miR-1NC or miR- 1mimic for 48 h. The relative levels of Frizzled 7, TNKS2 and c-Myc expression in MCF-7/miR-1NC. MCF-7/miR-1mimic, SKBR3/miR-1NC, SKBR3/miR-1mimic, untransfected MCF-7/CSC and SKBR3/CSC cells were determined by Western blot assays [10 (link)]. The primary antibodies included goat anti-Frizzled 7 (sc-31061), TNKS2 (SC-22854), mouse anti-c-Myc (sc-40) and rabbit anti-GAPDH (sc-25778, Santa Cruz Biotechnology, Santa Cruz, USA) and negative controls of rabbit or mouse IgG. The relative levels of each interesting protein to GAPDH were determined using the Gel pro4.0.
Similarly, the levels of Oct4 and Nanog as well as cytosol and nuclear β-catenin to control GAPDH or Lamin B1 (Santa Cruz Biotechnology) in MCF-7/CSC, MCF-7/miR-1NC, MCF-7/miR-1mimic, MCF-7/miR-1inhibitor, SKBR3/CSC, SKBR3/miR-1NC, SKBR3/miR-1mimic, SKBR3/miR-1inhibitor CSCs were characterized by Western blot using rabbit anti-Oct4 (sc-9081), anti-Nanog (sc-33759) and goat anti-Lamin B1 (sc-30264).