Horseradish peroxidase conjugated anti rabbit igg na931v

Proteins from cell lysates (20–60 μg), and tissue samples (20–70 µg) were separated on a 10% or 10–20% gradient acrylamide gel and then transferred to a 0.45 μM pore PVDF membrane (Merck, Darmstadt, Germany) as previously described [57 (link)]. The primary antibodies used are shown in Supplemental Table S4 and all of them were diluted in TTBS. Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated anti-rabbit IgG (NA931V; 1:4000 in TTBS) or anti-mouse IgG secondary antibody (NA934V; 1:5000 in TTBS) (GE Healthcare, Buckinghamshire, UK), respectively. When possible, phospho-proteins and their total expression were detected in the same gel, using Restore^TM Western Blot Stripping Buffer (Thermo Fisher Scientific) as per the manufacturer’s instructions, blocking the membrane again before the incubation with the next antibody. Loading was normalized by β-actin or α-tubulin. Protein bands were visualized using the Clarity Western Blot Analysis ECL (BioRad^®, Hercules, CA, USA). Band densitometry was analysed using ImageJ Software (v1.52a, Wayne Rasband, National Institute of Health, Stapleton, NY, USA).

Proteins from cell lysates (20–40 μg), and tissue samples (60 μg) were separated on a 10% or 10%−20% gradient acrylamide gel and then transferred to a 0.45 μM pore PVDF membrane (Merck, Darmstadt, Germany) as previously described.¹⁶, ¹⁷ The primary antibodies used are shown in Table S2 and all of them were diluted in TTBS. Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase‐conjugated anti‐rabbit IgG (NA931V; 1:4000 in TTBS) or anti‐mouse IgG secondary antibody (NA934V; 1:5000 in TTBS) (GE Healthcare, Buckinghamshire, UK), respectively. When possible, phosphoproteins and their total expression were detected in the same gel, using Restore^™ Western Blot Stripping Buffer (Thermo Fisher Scientific) as per the manufacturer's instructions, blocking the membrane again before the incubation with the next antibody. Loading was normalized by β‐actin or α‐tubulin. Protein bands were visualized using the SuperSignal^™ West Pico PLUS Chemiluminescent Substrate (34580, Thermo Fisher Scientific^®, Hercules, CA, USA). Band densitometry was analysed using ImageJ Software (v1.52a, Wayne Rasband, National Institute of Health, Stapleton, USA).