Ab22851

Total proteins (30 μg) from tumor lysates were separated by 12% SDS-PAGE gel, followed by transferring to PVDF membranes (Millipore, Billerica, MA, USA). The primary antibodies KLF6 (1:300, 67,297–1, Proteintech, China), KLF15 (1:300, ab22851, Abcam, UK),and GAPDH (1:800, ab8245, Abcam, UK) were used to incubate with the membrane at 4 °C overnight. After incubation for 1 h with the HRP-conjugated secondary antibody, an ECL Kit (Millipore) and a UV Transilluminator (Bio-Rad) were used to acquire and analyze the protein band intensities.

Adult hearts with pericardial nephrocytes from 7-day-old females were dissected in artificial Drosophila haemolymph according to Selma-Soriano et al. (2018a) (link), fixed with 4% paraformaldehyde (PFA) in PBS for 20 min and permeabilized by washing three times with PBS containing 0.3% Triton X-100 (PBS-T) for 10 min. Then, hearts were blocked in PBS-T containing 0.5% bovine serum albumin for 30 min at room temperature and incubated with the corresponding primary antibody (1:100) overnight at 4°C. Primary antibodies used were anti-Rph (Abcam, ab3338; with 50% of homology), anti-Hrs (DSHB, AB 2722114), anti-Rab3 and anti-Rab27 (BD Bioscience, 610379 and 558532, respectively) and anti-Klf15 (Ivy et al., 2015 (link)) (Abcam, ab22851; with 59% of homology). After three washes with PBS-T, the secondary antibodies (1:200), AlexaFluor-647 donkey anti-rabbit (Life Technologies, A31573), anti-mouse biotinylated (Sigma-Aldrich, B7264) and anti-rabbit biotinylated (Sigma-Aldrich, B8895) were incubated for 2 h at room temperature. Hearts with pericardial nephrocytes were then incubated with ABC solution (ABC kit, VECTASTAIN) for 30 min at room temperature, followed by washes and incubation with streptavidin-Texas Red (Vector Laboratories, 1:500). All images were taken using an LSM 800 confocal microscope (Zeiss) and were processed using ZEN software.