Hrp conjugated mouse anti β actin

Cells were grown to ~70% confluency in DMEM including 10% Foetal Bovine Serum. The IP was carried out with rabbit polyclonal anti-MTDH (Abnova (H00092140-PW2), Dallas TX, USA) and performed as described previously^{59 (link)}. MTDH IP was valuated using Western blotting analysis, according to standard procedures. Briefly, gel electrophoresis and protein transfer were performed using XCell SureLock Mini-Cell with Blot Module™ (Invitrogen, Life Technologies). Membranes were blocked and incubated for at least 16 hours at room temperature with mouse polyclonal anti-MTHD (Abnova (H00092140-PW2), Dallas TX, USA; dilution 1:500). Following incubation with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, Glostrup, DK; dilution 1:10.000) proteins were detected using ECL prime Western Blotting Reagent (GE Healthcare, Chalfont St. Giles, UK) and ChemiDoc-It Imaging System (UVP, Upland, CA, USA). The β-actin was visualized using HRP-conjugated mouse anti-β-actin (Abcam; dilution 1:10.000).

Western blotting was performed as described previously (47 (link)). A total of 5 μg protein was mixed with 4× Laemmli sample buffer (Bio-Rad Laboratories) and boiled at 95°C for 5 minutes. Each protein sample was separated by SDS-PAGE in 4%–20% TGX-GEL (Bio-Rad Laboratories) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories) by using a semidry method. The membranes were blocked in 5% skim milk in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 1 hour with shaking at RT. Afterward, the membrane was incubated overnight with shaking at 4°C with primary antibodies, which cross both human and mice — rabbit anti–E-cadherin (1:1000, 24E10, Cell Signaling Technology), rabbit anti-vimentin (1:1000, Abcam), or rabbit anti-FAP (1:500, Abcam) — and then incubated with horseradish peroxidase–conjugated (HRP-conjugated) secondary antibodies for 1 hour with shaking at RT. Washes before and after antibody reactions were done on a shaker, 3 times within TBS-T for 10 minutes each at RT. Alternatively, before the detection of β-actin, we performed the stripping procedure for 10 minutes because the molecular weight of vimentin and β-actin are similar. Then, the membranes were incubated with HRP-conjugated mouse anti–β-actin (1:20,000, Abcam) antibody for 1 hour with shaking at RT. Blots were visualized with ECL substrate (Bio-Rad Laboratories).