Gfp b 2 sc 9996

Total protein was resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for Phospho-STAT3 (S727) (ab32143, abcam), Phospho-STAT3 (Y705) (9131, Cell Signalling Technology (CST)), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology), Phospho-STAT5 (Y694) (9314, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), involucrin (SY5, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (C1P5, Santa Cruz Biotechnology), HPV 16 E7 (ED17, Santa Cruz Biotechnology), Phospho-ERK1/2 (Thr202/Tyr204) (43705, CST), Phospho-JNK (Thr183/Tyr185) 4668, CST), Phospho-p38 (Thr180/Tyr182) (9211, CST), Bcl xL (H-62, Santa Cruz Biotechnology), Cyclin D1 (A-12, Santa Cruz Biotechnology) p53 (FL-393, Santa Cruz Biotechnology), p21 (2947, CST), FLAG (F3165, Sigma), GFP (B-2: sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology). Western blots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL (Thermo/Pierce).

To prepare cell lysates, log-phase cells (OD₆₀₀ 2.0) were pelleted, resuspended in sample buffer (2% SDS, 1% 2-mercaptoethanol), boiled and lysed by glass-bead mechanical disruption [7 (link)]. The lysates were collected after centrifugation. After separating the proteins by 10% SDS–PAGE, samples were transferred to nitrocellulose or PVDF blocked with 5% skimmed milk in TBS-T (137 mM NaCl, 15.2 mM Tris-HCl, 4.54 mM Tris, 0.896 mM Tween 20) and then probed with primary and fluorescent secondary antibodies. Mouse α-GFP antibody was from Santa Cruz Biotechnology (1 : 2000, GFP(B-2):sc-9996). Peroxidase conjugated α-mouse IgG (1 : 5000; Sigma, A-4416) treated with ECL (Thermo Scientific) was used to develop the western blot.

Antibodies used for immunoblotting in this study were as follows: polyclonal antibodies detecting NifU_Av, NifS_Av, NifB_Av and NifB_Mi were raised against purified preparations of the corresponding A. vinelandii or M. infernus proteins. Rubisco specific antibodies were a kind gift from Prof. Göran Samuelsson, Umeå University. His-tag (H-3, sc-8036, Santa Cruz), HA-tag (3F10, 12013819001, Roche), GFP (B-2, sc-9996, Santa Cruz), Strep-tag II (StrepMAB-Classic, 2-1507-001, IBA Lifesciences) specific antibodies are commercially available.