Goat anti rabbit igg hpr

After feeding the fruit flies with 100 μg/mL L. reuteri for five weeks, the total proteins from 50 male fly heads were lysed with a PRO-PREP protein extraction buffer (#17081, iNtRON Biotechnology, South Korea) and centrifuged for 10 minutes at 13,000 r/min. Three times were conducted independently, and two replicates per trial were tested. The supernatants of the samples were quantified using a Bradford assay, and an equal amount of protein was loaded. Western Blot was performed with rabbit polyclonal anti-Drosophila phospho-Ser505 AKT antibody (1:1000, #4054, Cell Signaling, USA) and rabbit monoclonal anti-Drosophila AKT antibody (1:1000, #9272, Cell Signaling, USA). Rabbit monoclonal anti-α tubulin antibody (1:10000, #SAB4500087, Sigma-Aldrich, USA) was used as the loading control. The goat anti-rabbit IgG-HPR (1:5000, #sc-2004, Santa Cruz, USA) was used as the secondary antibody. The phospho-Ser505 AKT/AKT ratio was quantified using ImageJ software. The data are presented as mean ± SEM values, and the statistical probability was determined using a t-test.

Harvested cells were lysed in RIPA buffer (150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5 and 2 mM EDTA) (GenDEPOT, TX, USA) containing 1% protease inhibitor cocktail (GenDEPOT). 20 μg of each sample is separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). Western blotting was performed as previously described by Liu et al.³⁸ with primary antibodies to mouse anti-E-cadherin 1:1000 (Cell Signalling, Danvers, MA, USA), rabbit anti-Vimentin 1:500 (Abcam, Cambridge, UK), rabbit anti-Zeb1 1:500 (Sigma), rabbit anti-IL-6 1:1000 (Abcam), rabbit anti-STAT3 1:500 (phosphor Y705: Abcam), mouse anti-STAT3 1:1000 (Cell signalling), rabbit anti-α-lamin 1:1000 and goat anti-β-actin 1:5000 (Santa Cruz Biotechnology, Santa Cruz, USA). All primary antibodies were diluted in 5% Bovine Serum Albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C. Secondary antibody included goat anti-rabbit IgG-HPR 1:5000 (Santa Cruz Biotechnology), goat anti-mouse IgG-HPR 1:2000 (Santa Cruz Biotechnology) and rabbit anti-goat IgG-HPR (GenDepot, TX, USA). Protein bands were visualised using enhanced chemiluminescence reagents (Western Lighting Plus, PerkinElmer, USA).