Goat anti rabbit 680rd

The following primary and secondary antibodies were applied for immunodetection and immunostaining: anti-ataxin-3 (1:5000; 1H9, MAB5360, Merck, Darmstadt, Germany), anti-ataxin-3/C-terminal (1:2500; SA3637) [20 (link)], anti-KPNB1 (1:5000; H-300, sc-11367, Santa Cruz Biotechnology, Heidelberg, Germany), anti-KPNA3 (1:5000; ab137446, Abcam, Cambridge, United Kingdom), anti-CLPP (1:2500; 15,698–1-AP, Proteintech, St. Leon-Rot, Germany), anti-GAPDH (1:5000; 0411, sc-47724, Santa Cruz Biotechnology), anti-β-actin (1:5000; AC-15, A5441, Sigma-Aldrich), anti-vinculin (1:1000; E1E9V, 13,901, Cell Signaling Technologies, Frankfurt am Main, Germany), anti-lamin A/C (1:5000; 346, sc-7293, Santa Cruz Biotechnology), anti-GST (1:2500; B-14, sc-138, Santa Cruz Biotechnology), anti-α-spectrin (1:1000; AA6, Merck), anti-GFP (1:1000; B-2, sc-9996, Santa Cruz Biotechnology), anti-LC3 (1:1000; MBL-PD014, MBL International, Woburn, MA, US), anti-p62 (1:1000; 5114, Cell Signaling Technologies), and IRDye secondary antibodies goat anti-mouse 680LT, goat anti-mouse 800CW, goat anti-rabbit 680RD, goat anti-rabbit 800CW (all 1:10,000; LI-COR Biosciences, Bad Homburg, Germany), and Alexa Fluor 555 (1:500; Thermo Fisher Scientific).

Cellular protein was extracted using Pierce IP Lysis Buffer (Thermo Scientific, Rockford, IL, USA) and a mixture of Halt Protease Inhibitor Cocktail 100X (Thermo Scientific, Rockford, IL, USA) and Phosphatase Inhibitor Cocktail 100× (Cell Signaling Technology, Danvers, MA, USA). Sixty micrograms of protein were applied to a 10% SDS/PAGE and transferred to nitrocellulose membranes. Membranes were incubated with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) prior to incubation with polyclonal or monoclonal antibodies to phospho-PKA substrates, PKA-Cα subunit, FasL, Fas, FADD, DR4, phospho-S15 TP53, total TP53, phospho-p38 MAPK, total p38 MAPK, phospho-S245 ATF4, total ATF4, BiP, and β-actin overnight at 4 °C. All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) with the exception of phospho-S245 ATF4 from Sigma Aldrich (St. Louis, MO, USA). Goat anti-rabbit IgG (H + L) 800 CW, goat anti-rabbit (680 RD) and/or goat anti-mouse (H + L) secondary antibodies were applied for 1 h at room temperature (1:25,000, LI-COR) prior to washing with 1× Tris Buffered Saline Tween-20 (TBS-T). Visualization was carried out with the LI-COR Odyssey CLx imaging system and software.

Strain AJY4406 was transformed separately with plasmids pAJ5125, pAJ4853, pAJ5402, pAJ5403 and pAJ5404. Transformants were grown to stationary phase, diluted 1:20 and grown for 5 h at 30 °C. Cells were harvested and proteins extracted by incubating in 100 mM NaOH for 20 min on ice, then boiling in sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) loading buffer for 5 min. Protein extracts were resolved on a 6–18% polyacrylamide gel, transferred to a nitrocellulose membrane and incubated overnight at 4 °C in tris-buffered saline (TBS) supplemented with 1% casein, containing primary antibodies rabbit anti-glucose-6-phosphate dehydrogenase (diluted 1:20,000) and rat anti-FLAG (Agilent, diluted 1:20,000). After two washes with TBS + Tween 20 (TBS-T) and one with TBS, the membrane was incubated for 30 min in TBS containing secondary antibodies goat anti-rabbit 680RD and goat anti-rat 800CW (LiCOR, each diluted 1:20,000). After three washes with TBS, secondary antibodies were visualized on a LiCOR Odyssey infrared scanner.

Calvaria were dissected from E15.5 WT embryos and tissue samples taken from osteogenic front of the frontal bone and from interfrontal midsutural mesenchyme. Samples were pooled from three calvaria of the same litter and this was repeated 4 times. Age-matched brain tissue was used as control. Western blotting was carried out as described previously (Tanimoto et al., 2012 (link)). Briefly, 10 μg of each sample was probed for GLI3 antibody (AF3690, R&D Systems), GLI1 antibody (2643, Cell Signaling Technology), α-tubulin (T6199, Sigma-Aldrich). 20 μg of total protein from siRNA treated calvarial cells was probed for RUNX2 antibody (8486, Cell Signaling Technology), IHH antibody (AF1705, R&D Systems), and normalized against signal by Actin antibody (A2066 Sigma), detected by secondary antibodies goat anti-mouse IRDye 800CW (926-32210, LI-COR) and goat anti-rabbit 680RD (926-68071, LI-COR). Blots were analyzed with an Odyssey CLx infrared imager (model 9120) (Li-Cor) and Image-J (NIH) software. Statistical values were calculated using Student's t-test, with P-value below 0.05 indicated as significant.