Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.

doi: 10.1073/pnas.1119296109

Figure Lengend Snippet: Fig. 1. TBK1 protects MEFs from TNF-induced apoptosis and modulates IKK-dependent phosphorylation of RelA/p65. (A) Wt and Tbk1−/−MEFs were treated for 24 h with TNF (25 ng/mL) alone or for 3 h with TNF in the presence of CHX (10 μg/mL). Apoptotic cells were detected by TUNEL assays. The bars represent averages ± SD of three different experiments. Approximately 1,200 cells were counted in each experiment. (B) MEFs treated with TNF and CHX for the indicated times were analyzed for caspase-8 and caspase-3 activation by immunodetection of their cleaved forms. (C) Cell extracts prepared from Tbk1−/−

Article Snippet: Antibodies against RelA/p65 (S536) (no. 3031), phospho-IκBα (no. 9241), phospho-MAPKs (no. 9910), phospho-cJun (no. 9261), ERK, p38 (no. 9212), TBK1 (no. 3012), Bcl-XL (no. 2764), c-IAP1 (no. 4952), mBID (no. 2003), caspase-3 (no. 9662), cleaved caspase-3 (no. 9661), and cleaved PARP (no. 9544) were from Cell Signaling; antibodies to IKKγ (no. 557383), JNK1 (no. 551196), XIAP (no. 610716), CD95/Fas receptor (554254), caspase-8 (no. 559932), and procaspase-3 (no. 65906E) were from BD Transduction Laboratories; antibodies to IKKα (no. IMG-136A), IKKβ (no. IMG-129A), TBK1 (IMG-139A), and IκBα (no. IMG-127A) were from Imgenex; antibodies against RelA (sc-372 and sc-372G), RelB, cRel, IκBβ, HSP60, and PAI-2 (sc-25746) were from Santa Cruz Biotechnology; anti-p65/RelA (CT), anti–phospho-ATF-2 (no. 05–891), anti-TG2 (no. 06–471), and anti-caspase-1 (no. 06–503) were from Upstate Biotechnology; anti–CREB-1 (AB3006) was from Millipore; anti-FLIPα (CT; no. 1161) was from ProSci, anti-TG2 (AB-4) was from Neomarkers (Lab Vision), anti-HA (clone 3F10) was from Roche, anti-cAIP2 was from R&D Systems, and anti-CD31 was from Abcam.

Techniques: Phospho-proteomics, TUNEL Assay, Activation Assay, Immunodetection

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator

doi: 10.1101/2023.06.06.543873

Figure Lengend Snippet: A. Schematic overview of IKK signaling. Canonical NF-κB is regulated by the IKK complex, which consists of the IKKα, β, and γ subunits. Phosphorylation of IκBα leads to its degradation, allowing the p65-p50 dimer to accumulate in the nucleus and regulate transcription of target genes. B. Relative mRNA levels of IKBKB, IKBKG and CHUK (normalized to TBP ) in IKBKB-KO , IKBKG-KO and CHUK-KO LNCaP cells (n=3, mean values are depicted and error bars represent the standard deviation). P-value was calculated using a two-way ANOVA test. C. Protein levels of IKBKB, IKBKG and CHUK protein levels in IKBKB-KO , IKBKG-KO and CHUK-KO LNCaP cells. Actin and HSP90 are used as loading control. Cas9 was used as a control for efficient lentiCRISPRv2 transfection. D. Crystal violet assay of NT control and IKBKB-KO , IKBKG-KO and CHUK-KO cells cultured as a monoculture (up) and co-culture (down) with macrophages. Cells were cultured for 3 days. Crystal violet staining was quantified using Fiji software.

Article Snippet: After transfer, membranes were stained with Ponceau S and subsequently blocked in 4% BSA (A8022, Sigma/Merck) in 1x PBS-Tween (137 mM NACl, 10 mM Na 2 HP04, 1.5 mM KH 2 PO 4 , 2.6 mM KCl, 0.1% Tween-20) for 1h and incubated with primary antibodies against IKKα (2682, Cell signaling, 1:1000), IKKβ (10AG2, Novus Biological, 1:000), IKKγ (2685, Cell signaling, 1:1000), PKCδ (2058, Cell signaling, 1:1000), Actin (MAB1501R, Merck, 1:1000), HSP90 (sc-12119, Santa Cruz Biotechnologies, 1:1000) diluted in 4% BSA/PBS-T. Blots were incubated overnight at 4°C and washed five times with PBS-T prior to incubation with secondary antibodies donkey-α-mouse 680 RD (926-68073, LI-COR Biosciences, 1:10000) and donkey-α-rabbit 800 CW (926-32213, LI-COR Biosciences, 1:10000), diluted in 4% BSA/PBS-T for 1h.

Techniques: Phospho-proteomics, Standard Deviation, Control, Transfection, Crystal Violet Assay, Cell Culture, Co-Culture Assay, Staining, Software