Journal: Nature Communications

Article Title: Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy

doi: 10.1038/s41467-023-41243-4

Figure Lengend Snippet: a Left panel: sequence comparison of hAtg3 C-terminal residues 262–277 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 265–277. b Representative SDS-PAGE gel images of time-dependent formation of LC3B-PE for hAtg3 wildtype (WT) and mutants ( n = 3). CL is the control incubated for 180 min without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. c Left panel: sequence comparison of hAtg3 C-terminal residues 291–300 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 291–300. d Representative SDS-PAGE gel images of time-dependent formation of LC3B-PE for hAtg3 mutants ( n = 3). CL is the control incubated for 180 min without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. For b and d , source data are provided as a Source Data file.

Article Snippet: Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1) (ThermoFisher scientific, J61835.MCR) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5000.

Techniques: Sequencing, Comparison, SDS Page, Control, Incubation, Liposomes

Journal: Nature Communications

Article Title: Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy

doi: 10.1038/s41467-023-41243-4

Figure Lengend Snippet: a Atg3 knockout (Atg3 −/− ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry-EV (empty vector), mCherry-hAtg3, mCherry-hAtg3 F296L , or mCherry-hAtg3 F296S were cultured in complete media with 100 nM bafilomycin A1 for 3 h and subjected to immunoblotting for mCherry. b Representative immunoblot ( n = 4 blots). MEF cells were cultured in complete media (CM) or starvation media (SM) with/without 100 nM bafilomycin A1 (BafA1) to block LC3B-II (LC3B-PE) degradation for 3 h and subjected to immunoblotting with indicated antibodies. c Quantitative analysis of the relative LC3B-II level ( n = 4 blots) in in vivo LC3B lipidation experiments. d Quantitative analysis of induced autophagic flux ( n = 4 blots). Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. In c , data are presented as mean ± SD, P values: **** P < 0.0001, *** P = 0.0001, 0.0002, 0.0001, 0.0001 (left to right, bottom to top); ** P = 0.0021, 0.0017, 0.002, 0.0052, and 0.0034 (left to right, bottom to top). In d , data are presented as mean ± SD, P values: * P = 0.0125, 0.0283, and 0.0118 (bottom to top), ** P = 0.0053. Source data are provided as a Source Data file.

Article Snippet: Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1) (ThermoFisher scientific, J61835.MCR) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5000.

Techniques: Knock-Out, Stable Transfection, Expressing, Plasmid Preparation, Cell Culture, Western Blot, Blocking Assay, In Vivo

Journal: Molecular Metabolism

Article Title: Loss of Tbk1 kinase activity protects mice from diet-induced metabolic dysfunction

doi: 10.1016/j.molmet.2018.06.007

Figure Lengend Snippet: Tbk1 Δ/ Δ mice have less inflammation relative to Tbk1 +/ + mice on HFD . (A) Representative 20x images and quantification of cd11b/c stained subcutaneous white adipose tissue from Tbk1 Δ/Δ and Tbk1 +/+ mice after 10 weeks on ND or HFD (n = 4–8 mice/group). Scale bar indicates 200 μM and ‘CLS’ refers to crown like structures. (B) mRNA expression of genes encoding CD11c, F4/80, TNFα, IL-6 and IL-12 in subcutaneous WAT of Tbk1 Δ/Δ and Tbk1 +/+ mice fed with HFD as indicated (n = 4–6 mice/group). (C) Liver tissue lysates from 14-week-old HFD-fed Tbk1 Δ/Δ and Tbk1 +/+ mice were immunoblotted with antibodies against IL-6 and IL-1β. β-actin and GAPDH were used as internal loading controls. Indicated cytokines from WAT (D) and liver tissue (E) lysates of HFD-fed Tbk1 +/+ and Tbk1 Δ/Δ mice were measured by Bio-Rad multiplex array (n = 4–8 mice/group for WAT, n = 8–12 mice/group for liver). All mice are from 129S5 background. Results are representative of mean +/− SEM. Statistical analysis by Student's t -test. * p < 0.05, ** p < 0.01.

Article Snippet: Sections for immunohistochemical analysis were blocked with 20% aquablock and incubated with rabbit anti-cd11b/c (Novus Biologicals, NB110-40766) in blocking solution (5% BSA in TBS with 0.05% tween) at 4 °C overnight.

Techniques: Staining, Expressing, Multiplex Assay

Journal: BMC Cancer

Article Title: Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

doi: 10.1186/1471-2407-10-379

Figure Lengend Snippet: Gene-silencing of Bcl-2, Survivin and XIAP . Western blot showed efficient silencing in transfected MiaPaCa-2 ( A ) and AsPC-1 cells ( B ). 50,000 cells/well were single-transfected with carrier solution (lane 1) and siRNA against Luciferase (lane 2) as control. SGS, lowST and ST all effectively silenced the three target genes (lane 3-5). Efficient knock-down was also shown in the lowST group by RT-PCR in MiaPaCa-2 ( C ) and AsPC-1 cells ( D ). White bars show controls, grey bars signify transfected cells. All samples were normalized to β-Actin as a house-keeping gene. SGS = Simultaneous gene silencing; lowST = Low dose siRNA transfection; ST = Standard dose siRNA transfection.

Article Snippet: Subsequently, the membrane was either incubated with an antibody to Survivin (1:1000, NB 500-201, Novus Biologicals, Acris Antibodies, Hiddenhausen, Germany), Bcl-2 (1:200, sc-509, Santa Cruz, Heidelberg, Germany), XIAP (1:500, 610716, Becton Dickinson, Heidelberg, Germany) or GAPDH (1:5000, ab 8245-100, abcam, Cambridge, UK) in TBS-T 0.1% with blocking as mentioned above for 1 h. After washing in TBS, the protein was visualized using the ECL detection kit (GE Healthcare, Munich, Germany) with a peroxidase-labeled anti-mouse antibody (1:25,000, NIF825, GE Healthcare, Munich, Germany) or an anti-rabbit antibody (1:5,000, NIF824, GE Healthcare, Munich, Germany).

Techniques: Western Blot, Transfection, Luciferase, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction