Transcription factor 2 b tfiib

Cytosolic protein was isolated from cells in lysis buffer A, containing Hepes (10 mM), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1 mM), PMSF (0.5 mM), Protease Inhibitor Cocktail (15 µL/mL; Sigma-Aldrich), Igepal CA-630 (0.5%; Sigma-Aldrich), pH 7.9. The suspension was centrifuged, and the supernatant (cytosolic extract) was stored. Nuclear extracts were obtained by incubating the pellet obtained from the cytosolic extract in lysis buffer B, containing HEPES (20 mM), NaCl (0.4 M), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (0.5 mM), Protease Inhibitor Cocktail (15 µL/mL), pH 7.9. Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). To determine specific protein contents, 20 µg of nuclear cell lysates were analyzed by immunoblotting using antibodies against proliferating cell nuclear antigen (PCNA; 1:100; Santa Cruz Biotechnology INC, Dallas, TX, USA), Cyclin D1 (1:500; Cell Signaling Technology Inc., Danvers, MA, USA) and cleaved Notch 1 (Val1744) (1:200; Cell Signaling Technology) as primary antibodies, and horseradish peroxidase-conjugated goat anti-mouse (1:10000; Santa Cruz Biotechnology) and goat anti-rabbit (1:10000; Santa Cruz Biotechnology) as secondary antibodies. Transcription factor II B (TFIIB; 1:1000; Cell Signaling Technology) was used as loading control.

Western blot analysis for erythropoietin (EPO) (1:500, Abcam), STAT5a,b (1:600), and Bcl-XL (1:1000), SAPK/JNK (1:1000, Cell Signaling), and phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (1:11,000, Cell Signaling) was performed using 19 μg protein extract from naïve mice and mice with 1 and 6 h survival after SCI. Proteins were separated on 4-12% SDS-PAGE gels (Invitrogen) using MOPS SDS (Invitrogen) containing 0.25% antioxidant (Invitrogen) as previously described [13 (link)]. β-actin (1:100,000, Sigma-Aldrich) or transcription factor II B (TFIIB) (1:1000, Cell Signaling, Leiden, The Netherlands) were used as loading controls. SeeBlue Plus2 Prestained standard (Invitrogen) was used as a molecular marker. Bands were quantified using Image Lab Software (Bio-Rad, Copenhagen, Denmark).
The same naïve Tnf^fl/fl mice were included on all gels and data were normalized according to protein concentrations in individual plots. Analysis was performed on unmerged blots with the same exposure time using Image Lab (Bio-Rad). Analysis was performed with n = 5 mice/group and data were normalized to the loading control and presented as percentages relative to naïve Tnf^fl/fl mice.

Western blotting was performed on one series of sections as described in Clausen et al. [9 (link)] with minor modifications. Western blotting for phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (1:1000), p-p44/p42 MAPK (ERK1/2)(1:2000) and p-p38 MAPK (Tyr180/Tyr182) (1:1000) (S9910 Kit, Cell Signaling) was performed using 4–12 % gels (Life Technologies) using transcription factor IIB (TFIIB) (Cell Signaling, 1:1000) as a loading control and SeeBlue Plus2 standard (Invitrogen) as an indicator of size. Bands were amplified using a SuperSignal^®Extended duration substrate (34075, Thermo) and visualized by chemiluminescence using the FUSION 7X camera. Densitometry was performed using Image J (version 1.47v) (NIH) following recommendations of the Image J developers. Analysis was performed on two independent gels with n = 2/group and data were normalized to TFIIB and represented as percentages relative to naive LM mice.