lsm1 rabbit  (Absolute Biotech Inc)

Journal: Virology

Article Title: Processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection.

doi: 10.1016/j.virol.2014.04.022

Figure Lengend Snippet: Fig. 2. Various PB markers accumulated in the HFF cells infected with HCMV. (A) The amount of different PB markers increased during HCMV infection. HFF cells were infected with HCMV at an moi of 3 in the absence or presence of 200 μM cycloheximide and then harvested at 0, 6, 24, and 48 hpi in the absence of cycloheximide and 0 and 24 hpi in the presence of 200 μM cycloheximide. Immunoblot analysis was performed using an antibody against HCMV IE1 (IE72) and IE2 (IE86), DCP1a, EDC4, DDX6, 4E T, Lsm1, Lsm14A, or GAPDH at the times indicated after HCMV infection. GAPDH served as a loading control. (B) Accumulation of various PB markers during HCMV infection. HFF cells were treated with 0.5 mM Arsenite for 30 min or infected with HCMV at an moi of 3 in the absence or presence of 200 μM cycloheximide and fixed at 0 and 24 hpi. The cells were stained with mouse monoclonal or rabbit polyclonal antibody specific for Dcp1a or Lsm14A, respectively, and goat polyclonal antibody specific for EDC4, followed by staining with Alexa 568-conjugated donkey anti- mouse or anti-rabbit IgG, respectively, and Alexa 680-conjugated anti-goat secondary antibodies. Histogram shows the ratio of PBs/cell counted using Dcp1a and EDC4 PB markers. Left and right bars represent the number of PBs with a diameter of less or more than 1 μm, respectively. nn indicates values of po0.01. (C) HFF cells were infected with HCMV at an moi of 3, fixed at 0 and 24 hpi, and then stained with mouse monoclonal antibody specific for Ago2 and goat polyclonal antibody specific for EDC4, followed by staining with Alexa 568- conjugated donkey anti-mouse IgG and Alexa 680-conjugated anti-goat secondary antibodies.

Article Snippet: Goat polyclonal antibody against EDC4 (sc-137444), and rabbit polyclonal antibody against Lsm1 (sc-67365) were from Santa Cruz Biotechnology, (Santa Cruz, CA) and mouse monoclonal antibody against Dcp1a, clone 3G4 (Cat# H00055802-M6), was from Abnova (Taipei, Taiwan).

Techniques: Infection, Western Blot, Control, Staining

Journal: Virology

Article Title: Processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection.

doi: 10.1016/j.virol.2014.04.022

Figure Lengend Snippet: Fig. 4. PB accumulation in HFF cells infected with HCMV or UV-inactivated HCMV. HFF cells were infected with HCMV (A or C), or UV-inactivated virions (B and D), or the culture fluid after removal of HCMV virions (E) at an moi of 3. HFF cells were maintained in the absence (A and B) or presence (C and D) of cycloheximide. Cells were analyzed for IE1 RNA by FISH and then stained with rabbit polyclonal antibody specific for Lsm14A and goat polyclonal antibody specific for EDC4, followed by staining with Alexa 488-conjugated donkey anti-rabbit and Alexa 680-conjugated anti-goat IgG secondary antibodies. (F) Histogram showing the number of PBs/cell. Thirty cells were selected from multiple fields of view, and the number of Lsm14A and EDC4 double-positive PBs were counted. Data are averages of merged dots determined by counting three times independently, and the error bars indicate SD.

Article Snippet: Goat polyclonal antibody against EDC4 (sc-137444), and rabbit polyclonal antibody against Lsm1 (sc-67365) were from Santa Cruz Biotechnology, (Santa Cruz, CA) and mouse monoclonal antibody against Dcp1a, clone 3G4 (Cat# H00055802-M6), was from Abnova (Taipei, Taiwan).

Techniques: Infection, Staining

Journal: Virology

Article Title: Processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection.

doi: 10.1016/j.virol.2014.04.022

Figure Lengend Snippet: Fig. 5. Cytoplasmic distributions of IE1 mRNA and PB markers, Lsm14A and EDC4. HFF cells were infected with HCMV at an moi of 3 and fixed at 1 (A), 3 (B), 6 (C), and 24 (D) hpi. Infected HFF cells were also maintained in the presence of cycloheximide and fixed at 24 hpi (E). These cells were analyzed with IE1 probe by FISH and then stained with rabbit polyclonal antibody specific for Lsm14A and goat polyclonal antibody specific for EDC4, followed by staining with Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 680-conjugated anti-goat secondary antibodies.

Article Snippet: Goat polyclonal antibody against EDC4 (sc-137444), and rabbit polyclonal antibody against Lsm1 (sc-67365) were from Santa Cruz Biotechnology, (Santa Cruz, CA) and mouse monoclonal antibody against Dcp1a, clone 3G4 (Cat# H00055802-M6), was from Abnova (Taipei, Taiwan).

Techniques: Infection, Staining

Journal: Virology

Article Title: Processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection.

doi: 10.1016/j.virol.2014.04.022

Figure Lengend Snippet: Fig. 6. Knockdown effect of PB components on viral gene expression in HCMV-infected cells. HFF cells were transfected with siRNAs targeting 4E-T, Lsm1, and LSM14A, or with a non-targeting siRNA (siNeg), and, 2 days after transfection, infected with HCMV at an moi of 3. (A) Effect of siRNAs specific for the PB components, 4E-T, Lsm1, and Lsm14A on mRNA expression of the corresponding gene and viral mRNA expression. The transfected and infected cells were harvested at 0, 3, 6, and 24 hpi. Total RNA was isolated and subjected to quantitative real-time RT-PCR analysis to detect the gene transcripts indicated on the Y axis. RNAs were normalized to HPRT RNA and values were calculated relative to siNeg at 0 (upper panel) or 3 (lower panel) hpi. Data are averages of three independent experiments. Statistical analyses were done using the t-test. RNA expression levels compared to siNeg were assessed for statistical significance. n indicates values of po0.05. nn indicates values of po0.01. (B) The transfected and infected cells were fixed at 24 hpi and stained with rabbit polyclonal antibody specific for Lsm14A and goat polyclonal antibody specific for EDC4, followed by staining with Alexa 568-conjugated donkey anti-rabbit IgG and Alexa 680-conjugated anti-goat secondary antibodies. Histogram showing the effect of siRNAs targeting 4E-T, Lsm1, and LSM14A, or with a non-targeting siRNA (siNeg) on the ratio of the PB number/cell. Thirty cells were selected from multiple fields of view, and the number of Lsm14A and EDC4 double- positive PBs were counted. Data are averages of merged dots determined by counting three times independently, and the error bars indicate SD.

Article Snippet: Goat polyclonal antibody against EDC4 (sc-137444), and rabbit polyclonal antibody against Lsm1 (sc-67365) were from Santa Cruz Biotechnology, (Santa Cruz, CA) and mouse monoclonal antibody against Dcp1a, clone 3G4 (Cat# H00055802-M6), was from Abnova (Taipei, Taiwan).

Techniques: Knockdown, Gene Expression, Infection, Transfection, Expressing, Isolation, Quantitative RT-PCR, RNA Expression, Staining