Journal: Advanced Science

Article Title: API5 Phosphorylation Promotes Antiviral Immunity by Inhibiting Degradation of Cytosolic RNA Sensor RLRs

doi: 10.1002/advs.202505479

Figure Lengend Snippet: API5 inhibits autophagic degradation of RIG‐I and MDA5 by phosphorylation. A and B) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected WT and API5 −/− A549 cells (A), and lungs from shCON and sh Api5 mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) or VSV (10 8.0 PFU mL −1 ; 60 µL per mouse) for 4 days (B). C and D) Immunoblot analysis of RIG‐I and MDA5 in SeV‐, VSV‐, and IAV‐infected API5 −/− A549 cells reintroduced with empty vector (Vec.), API5 WT , or API5 S464A (C), and the lung tissues from Flag‐tagged API5 WT ‐, API5 S464A ‐ or empty vector (Vec.)‐overexpressing mice infected with IAV (5×10 5 PFU mL −1 ; 50 µL per mouse) for 4 days (D). E) Immunoblot analysis of extracts of API5 −/− A549 cells treated with bafilomycin A1 (BafA1; 200 n m ), chloroquine (CQ; 50 µM), 3‐methyladenine (3‐MA; 10 m m ), MG132 (10 µM) or Lactacystin (Lacta; 10 µ m ) for 6 h followed by infected with SeV for 12 h. (F) Immunoblot analysis of extracts of WT, API5 −/− , ATG5 −/− , API5 −/− ATG5 −/‐ HEK293T cells infected with SeV for 12 h. G) Immunoblot analysis of extracts of WT and ATG5 −/− HEK293T cells transfected with empty vector (Vec.) or plasmid expressing Flag‐API 5WT , or Flag‐API5 S464A followed by SeV infection for 12 h. Data are one representative of three biological replicates.

Article Snippet: The following antibodies were used in this study: Rabbit anti‐Flag Tag (0912), Rabbit anti‐His Tag (0812), Mouse anti‐GST Tag (EM80701), Mouse anti‐β‐Actin (M1210), Mouse anti‐Histone H3 (M1306), Rabbit anti‐IKKα/β (ET1611‐23), Rabbit anti‐p65 (ET1603‐12), and Rabbit anti‐ATG5 (ET1611‐38) were purchased by HuaAn Biotechnology; Mouse anti‐Flag Tag (F1804) was from Sigma–Aldrich; Rabbit anti‐GAPDH (AB‐P‐R001) was from GoodHere Technology; Mouse anti‐influenza viral proteins were prepared and stored in the laboratory; Rabbit anti‐API5 (ab65836) and Rabbit anti‐p62 (ab109012) were from Abcam; Mouse anti‐API5 (sc‐393341) and Mouse anti‐RIG‐I (sc‐376845) were from Santa Cruz; Rabbit anti‐SRPK1(A5854) and Rabbit anti‐MDA5 (A13645) were from ABclonal; Rabbit anti‐ RIG‐I (67556‐1‐Ig) and Rabbit anti‐IRF3 (11312‐1‐AP) were from Proteintech; Rabbit anti‐phospho‐IRF3 (13 786) was from Signalway Antibody; Rabbit anti‐MDA5 (5321), Mouse anti‐Ubiquitin (3936), Rabbit anti‐TBK1(3504), Rabbit anti‐phospho‐TBK1 (5483), Rabbit anti‐phospho‐IKKα/β (2697), Rabbit anti‐phospho‐p65 (3033), Rabbit anti‐phospho‐IκBα (2859) and Rabbit anti‐LC3B (2775) were purchased by Cell Signaling Technology; HRP‐conjugated goat‐anti mouse IgG (074‐1806) and HRP‐conjugated goat‐anti rabbit IgG (074‐1506) were from Kirkegaard & Perry Laboratories, Inc. Bafilomycin A1(BafA1; HY‐100558), chloroquine (CQ; HY‐17589A), 3‐methyladenine (3‐MA; HY‐19312), MG132 (HY‐13259), Lactacystin (Lacta; HY‐16594), SRPIN340 (HY‐13949,) and cycloheximide (CHX; HY‐12320) were purchased from MedChemExpress.

Techniques: Phospho-proteomics, Western Blot, Infection, Plasmid Preparation, Transfection, Expressing

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells

doi: 10.4161/cc.7.7.5625

Figure Lengend Snippet: Percent of polymerized tubulin in cell lines treated with proteasome inhibitors

Article Snippet: Bortezomib [Velcade ® Millenium Pharmaceuticals, Cambridge, MA], MG-132 (Calbiochem, San Diego, CA), Epoxomycin, 15 and Z-Ile-Glu-(OtBu)-Alu-LeuH(aldehyde)(PSI) 16 (Peptides International, Osaka, Japan) and lactacystin (Peptides International or Calbiochem, San Diego, CA) were each reconstituted and stored according to the manufacturers’ instructions.

Techniques: Concentration Assay, Significance Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells

doi: 10.4161/cc.7.7.5625

Figure Lengend Snippet: Cell cycle distribution of cells treated with proteasome inhibitors for 24 hrs

Article Snippet: Bortezomib [Velcade ® Millenium Pharmaceuticals, Cambridge, MA], MG-132 (Calbiochem, San Diego, CA), Epoxomycin, 15 and Z-Ile-Glu-(OtBu)-Alu-LeuH(aldehyde)(PSI) 16 (Peptides International, Osaka, Japan) and lactacystin (Peptides International or Calbiochem, San Diego, CA) were each reconstituted and stored according to the manufacturers’ instructions.

Techniques:

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells

doi: 10.4161/cc.7.7.5625

Figure Lengend Snippet: Average fold Increase in acetylated α-tubulin in HCN2 cells treated with proteasome inhibitors

Article Snippet: Bortezomib [Velcade ® Millenium Pharmaceuticals, Cambridge, MA], MG-132 (Calbiochem, San Diego, CA), Epoxomycin, 15 and Z-Ile-Glu-(OtBu)-Alu-LeuH(aldehyde)(PSI) 16 (Peptides International, Osaka, Japan) and lactacystin (Peptides International or Calbiochem, San Diego, CA) were each reconstituted and stored according to the manufacturers’ instructions.

Techniques: Concentration Assay

Journal: PloS one

Article Title: Aberrant expression of interleukin-1β and inflammasome activation in human malignant gliomas.

doi: 10.1371/journal.pone.0103432

Figure Lengend Snippet: Figure 6. miR-132, miR-212 or the proteasome inhibitor lactacystin do not affect the expression of IL-1 in human astrocytes. Human astrocytes were transfected with specific or control anti-miR inhibitors (10 nM) for 48 h, and then stimulated with IL-1a for 24 h. (A) The expression of miR-132 was quantified by TaqMan real-time RT-PCR. Specific anti-miRs but not control anti-miR suppress miR-132 expression. (B) The culture supernatants were examined for the presence of IL-1b protein by sensitive ELISA with a lower detection limit of 3.9 pg/ml. There was no detectable IL-1b protein production in any of the human astrocyte cultures examined. (C) The effect of the proteasome inhibitor lactacystin on astrocyte IL-1b was examined. Astrocytes were treated with lactacystin at indicated concentrations with or without IL-1a, then cell lysates were subjected to ELISA after 24 h. IL-1b protein was undetectable under any conditions. Mean 6 SD from triplicate cultures. doi:10.1371/journal.pone.0103432.g006

Article Snippet: Lactacystin was purchased from Santa Cruz Biotechnology and was added to culture 10 min prior to cell stimulation.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Life

Article Title: Structural Design and Synthesis of Novel Cyclic Peptide Inhibitors Targeting Mycobacterium tuberculosis Transcription

doi: 10.3390/life12091333

Figure Lengend Snippet: Synthetic scheme of (Cyclo-1,6)Ac-CYYQWC-NH 2 with Rink amide MBHA resin: ( a ) Fmoc deprotection: piperidine 20% in DMF; ( b ) addition of Fmoc-L-Cys(Trt)-OH and Fmoc-L-Trp(Boc)-OH. Fmoc-L-Gln(Trt)-OH, Fmoc-L-Tyr(tBu)-OH (2×), Fmoc-L-Cys(Trt)-OH with HCTU/NMM in DMF as the coupling reagent; ( c ) final deprotection with 20% piperidine in DMF, followed by acetylation to the N-terminus with acetic anhydride and 2.6-lutidine in DMF, RT; ( d ) resin cleavage and global deprotection: TFA/TIPS/Phenol/H 2 O (90:5:2.5:2.5), RT, 2 h reaction, followed by crude precipitation and RP-HPLC purification resulted in the linear-Ac-CYYQWC-NH 2 ; and ( e ) cyclization with air dilution in a high dilution of ammonium bicarbonate buffer pH = 8.5, followed by characterization.

Article Snippet: A high-loading Rink amide methylbenzhydrylamine (MBHA) resin, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Phe-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, O-(1H-6-Chlorobenzotriazole-1-yl) (HCTU), and N-methyl morpholine (NMM) were purchased from Gyros Protein Technologies, Tucson, AZ, USA.

Techniques: Purification

Journal:

Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7

doi: 10.1128/JVI.75.16.7583-7591.2001

Figure Lengend Snippet: HPV-16 E7 degradation and E7-mediated pRB degradation are 26S proteasome-dependent but separable processes. (A) The human osteosarcoma cell line SAOS2 was transiently transfected with the indicated combinations of plasmids encoding wild-type pRB and wild-type HPV-16 E7 (16E7). Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB levels, normalized for GFP expression, is shown underneath those panels (in arbitrary units based on densitometry). (B) HPV-16 E7, adenovirus E1A, and SV40 T antigen oncoproteins differ in their ability to destabilize pRB. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7, adenovirus (Ad) E1A, and SV40 T antigen (TAg) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were processed for immunoblot analysis for pRB (upper panel) and cotransfected GFP control (lower panel). Quantitation of pRB (RB) levels, normalized for GFP expression, is shown underneath these panels. (C) Effect of MG132 and Lactacystin on HPV-16 E7 and pRB steady-state levels. SAOS2 cells were transfected with expression plasmids encoding pRB alone or in combination with 16E7. At 40 h posttransfection, cells were treated with 10 or 40 μM MG132 or Lactacystin or mock treated with dimethyl sulfoxide (DMSO) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (D) Effect of proteasome inhibition on various HPV E7 proteins. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7 (16E7 and 16E7-HA), a pRB-binding- and pRB degradation-deficient 16E7 mutant (16E7 ΔD21-C24), pRB-binding-competent and pRB degradation-deficient versions of E7 (16E7 ΔP6-E10 and 1aE7-HA), and a pRB-binding- and pRB degradation-competent 16E7 mutant (16E7 C91S) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (D) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 or HPV-16 E7 tagged with HA (middle panel), and cotransfected GFP control or HPV-1a E7 tagged with HA (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (E) E7 degradation and E7-mediated pRB degradation are not linked. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7 (16E7), a lysineless mutant of 16E7 (16E7 K60,97R), an amino-terminal FLAG-tagged version of 16E7 (FLAG-16E7), and a carboxyl-terminal FLAG-tagged version of 16E7 (16E7-FLAG) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (D) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (F) HPV-16 E7 stability correlates with E7-mediated pRB degradation. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding pRB and amino- and carboxyl-terminal FLAG-tagged HPV-16 E7. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and E7 levels, normalized for GFP expression, is shown underneath these panels.

Article Snippet: At 40 h posttransfection, cells were treated with either 10 or 40 μM MG132 or Lactacystin (BIOMOL, Plymouth Meeting, Pa.) for 4 h. Cells were then harvested by scraping into boiling lysis buffer containing 100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol (DTT), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 10% β-mercaptoethanol.

Techniques: Transfection, SDS Page, Western Blot, Quantitation Assay, Expressing, Plasmid Preparation, Inhibition, Binding Assay, Mutagenesis