Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: Elongator complex loss reduces SARS-CoV-2 and ZIKV infection. ( A ) Schematic representation of U34 tRNA modification pathway in FD (ELP1-deficient) cells. The Western blot shows ACE2 receptor and actin levels in cells transduced by ACE2-expressing lentivector (VLP ACE2 ). ( B ) Quantification of U34 modification levels (ncm 5 U, mcm 5 U, and mcm 5 s 2 U) in wild-type ( wt ) versus FD cells determined by mass spectrometry on tRNA-enriched RNA fractionations. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR in wt and FD cells at different multiplicities of infection (MOI) 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR in wt and FD cells at different MOIs 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Infection, Modification, Western Blot, Expressing, Mass Spectrometry, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: ALKBH8 and CTU1 knockdown individually impairs SARS-CoV-2 and ZIKV infection. ( A ) Schematic showing shRNA-mediated knockdown approach targeting U34 ALKBH8 and CTU1 components of U34 modification pathway. The Western blot analysis of ACE2 expression in A549 cells with and without lentiviral vector expressing ACE2 (VLP ACE2 ). ( B ) Western blot validation of ALKBH8 and CTU1 knockdown efficiency with corresponding actin loading controls. Numbers indicate relative protein levels. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR following knockdown of the indicated genes 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR following knockdown of the indicated genes 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Knockdown, Infection, shRNA, Modification, Western Blot, Expressing, Plasmid Preparation, Biomarker Discovery, Quantitative RT-PCR

Journal: Cell cycle (Georgetown, Tex.)

Article Title: IKKalpha regulates the mitotic phase of the cell cycle by modulating Aurora A phosphorylation.

doi: 10.4161/cc.5.20.3359

Figure Lengend Snippet: Figure 3. IKKa regulates the protein expression and phosphorylation of certain M phase regulators. (A&B) HeLa cells were transfected with control siRNA (lane 1) and siRNA targeting IKKa or Aurora B (lane 2) and IKKb or Aurora A (lane 3) for 48 hr. Cell lysates prepared from siRNA transfected HeLa cells were analyzed by Western blot for the expression of IKKa or Aurora B (first panel), IKKb or Aurora A (second panel), Plk1 (third panel), cyclin B1 (fourth panel), phospho‑histone H3 (fifth and sixth panels), histone H3 (seven panel) and actin (eight panel). (C) HeLa cells were transfected with control siRNA (lanes 1, 4, 7 and 10) and siRNAs targeting IKKa (lanes 2, 5, 8 and 11) and IKKb (lanes 3, 6, 9 and 12) followed by double thymidine treatment. After double thymidine treatment, HeLa cells were released and cell lysates were prepared at the indicated times. Cell lysates were analyzed by Western blot analysis for IKKa, IKKb, Plk1, cyclin B1, phospho‑H3 (S10), phospho‑H3 (S28), Aurora A, phospho‑Aurora A (T288), histone H3 and actin.

Article Snippet: The antibodies used were anti-IKKα (sc-7182), anti-Plk1 (sc-372), anti-phospho(S28) histone H3 (sc-12927), normal rabbit IgG (sc-2027), from Santa Cruz Biotechnology; anti -phospho (S10) histone H3 (05-598) and anti-cyclin B1 from Upstate Biotechnology; anti-IKKα (556532) and anti-Aurora B (611082) from BD Pharmingen; anti-Aurora A (3092), anti-phospho(T288) Aurora A (3091), anti-histone H3 (9715) from Cell Signaling.

Techniques: Expressing, Phospho-proteomics, Transfection, Control, Western Blot