Anti rabbit irdye 680 cw

Proteins were extracted using RIPA Lysis buffer containing Phosphatase Inhibitor Cocktail (Sigma Aldrich, Sydney, Australia) according to the manufacturer’s instructions. Protein concentration was quantified using a BCA Protein Determination Kit (Pierce) as per the manufacturer’s instructions and absorbance read at 562 nm on a PerkinElmer VICTOR X Multilabel Plate Reader. Western blots were performed as previously described [19 (link)], utilizing rabbit antibodies against phospho-STAT3 (Tyr 705), total-STAT3, BCL2 and ICAM-1 (Cell Signaling, Beverly, MA, USA), and a mouse monoclonal antibody against GAPDH (Millipore Technologies, Sydney, Australia) at 1:1000 dilution, followed by anti-rabbit IRDye™ 680 CW and anti-mouse IRDye™ 800 CW (LI-COR Biosciences, Nebraska, USA) at 1:10000 dilution in 5% (w/v) BSA in TBS containing 0.1% Tween. Images were obtained using an Odyssey Imaging System (LI-COR Biosciences) and analysed with ImageJ (NIH).

Cells were collected after treatment and lysed in a buffer of 50 nM Tris-HCl, pH 8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.5% Triton X-100 and 1% SDS and freshly supplemented with protease inhibitors (Roche, 11873580001). Then, 15 μg whole lysates per lane and a Chameleon Duo pre-stained protein ladder (LI-COR, 928-60000) were resolved on 4–12% Bis-Tris gels (NuPAGE Invitrogen, NP0321BOX) and transferred to Amersham Protran 0.2-μm nitrocellulose membranes (GE Healthcare, 10600001). Blots were blocked with a LI-COR blocking buffer and incubated with the primary antibodies, namely anti-FTH1 (3998S, Cell Signaling), anti-β-actin (A5441, Sigma), anti-CD71/TfR (Cell Signaling, D7S5Z, 1:1,000 dilution), anti-ZIP14 (PA5-21077, Thermo Fisher, 1:1,000 dilution) and anti-GAPDH (ab9485, Abcam, 1:2,000 dilution) at 4 °C overnight and subsequently incubated with the corresponding secondary anti-IgG antibodies, anti-rabbit IRDye 680 CW (1:15,000 dilution, LI-COR, 926-68071), anti-rabbit IRDye 800 CW (1:15,000 dilution, LI-COR, 926-32211) and anti-goat IRDye 680 CW (1:10,000 dilution, LI-COR, 926-68074) for 1 h at room temperature. Blots were analyzed with an Odyssey Fc imaging system (LI-COR). For densitometry, OD of the protein of interest relative to the housekeeping gene was normalized to the averages obtained in control samples.

Cells were harvested after treatment with the indicated compounds in NET buffer (150 mM NaCl, 50 mM Tris pH 8, 2 mM EDTA, 1% Triton, 0.1% FBS). Identical amounts of whole lysates (30 μg) together with a Chameleon™ Duo pre‐stained protein ladder (LI‐COR, #928‐60000) were resolved in 4–12% Bis‐Tris gels (NuPAGE Invitrogen, #NP0321BOX) and transferred to Amersham Protran 0.2 μm nitrocellulose membranes (GE Healthcare, #10600001). Blots were incubated with the primary antibodies rabbit anti‐P‐Rb S887/811 (1:1,000, Cell Signaling, #8516), rabbit anti‐FoxM1 D1205 (1:5,000, Cell Signaling, #5436), rabbit anti‐ACTB GTU‐88 (1:10,000, Sigma, #T6557), and goat anti‐p21 C‐19‐G (1:200, Santa Cruz, #sc‐397‐G) at 4°C overnight, and subsequently incubated with the corresponding secondary anti‐IgG antibodies anti‐rabbit IRDye 680 CW (1:15,000, LI‐COR, #926‐68071), anti‐rabbit IRDye 800 CW (1:15,000, LI‐COR, #926‐32211), and anti‐goat IRDye 680 CW (1:10,000, LI‐COR, #926‐68074) for 1 h at room temperature. Blots were analyzed with an Odyssey Fc imaging system (LI‐COR).