rabbit anti v5 pab  (Proteintech)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

doi: 10.1007/s00018-026-06146-9

Figure Lengend Snippet: ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with mouse anti-Flag mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments

Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Purification, Staining

Journal: Biochemical Journal

Article Title: JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

doi: 10.1042/BCJ20243020

Figure Lengend Snippet: ( A ) Schematic representation of predicted YBX1 interaction region (YIR) in HOTAIR promoter; ( B ) western blot of JMJD6 and YBX1 in parental MCF7, MDA MB 231, Vec and JOE cells. Numbers below western panels from Vec and JOE cells indicate densitometric scan results of three independent experiments. Mean fold change in protein amounts between these two cell lines is shown. ( C ) Immunofluorescent detection of JMJD6 and YBX1 in MDA MB 231, Vec and JOE cells. Nuclear–cytoplasmic fractionation and immunoblots for JMJD6 and YBX1 in MDA MB 231 cells ( D ), Vec ( E ) and JOE ( F ) cells. C, cytoplasmic extract; N, nuclear extract; W, whole cell lysate.

Article Snippet: Antibodies used for Western blots were JMJD6 for total protein (endogenous and exogenous) (PSR-H7, Santacruz Biotechnology sc-28348, USA, 1:1000), V5 for exogenously expressed JMJD6-V5 (Thermo Scientific, U.S.A., R960-25, 1:5000), YBX1 (exogenous and endogenous) (Abcam, UK, ab-12148, 1:2000), Myc for exogenously expressed Myc-YBX1 (Sigma Aldrich, U.S.A., 05–419, 1:2000), β-Actin as internal control (Thermo Scientific, U.S.A., A2228, 1:5000), Lamin A/C (Abclonal, U.S.A., 1:5000, A0249) and β tubulin (Abclonal, U.S.A., 1:5000, AC008) for nuclear and cytoplasmic markers respectively.

Techniques: Western Blot, Fractionation

Journal: Biochemical Journal

Article Title: JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

doi: 10.1042/BCJ20243020

Figure Lengend Snippet: Expression levels of JMJD6 ( A ) and YBX1 (B) as quantified by quantitative RT-PCR following transient overexpression in MCF7 cells. ( C ) Western blots for both proteins in transfected cells. Endogenous JMJD6 is marked by filled square and empty square indicates V5-tagged protein. ( D ) Densitometric scans of western blots from three independent experiments. ( E ) and (F) Expression of JMJD6 and YBX1 following siRNA mediated knock down in MCF7 cells. Expression levels in scrambled siRNA (si_Scr) treated cells were normalized to 1. ( G ) Western blots for both proteins in siRNA-treated cells. ( H ) Densitometric scans of immunoblots from three independent siRNA treatments.

Article Snippet: Antibodies used for Western blots were JMJD6 for total protein (endogenous and exogenous) (PSR-H7, Santacruz Biotechnology sc-28348, USA, 1:1000), V5 for exogenously expressed JMJD6-V5 (Thermo Scientific, U.S.A., R960-25, 1:5000), YBX1 (exogenous and endogenous) (Abcam, UK, ab-12148, 1:2000), Myc for exogenously expressed Myc-YBX1 (Sigma Aldrich, U.S.A., 05–419, 1:2000), β-Actin as internal control (Thermo Scientific, U.S.A., A2228, 1:5000), Lamin A/C (Abclonal, U.S.A., 1:5000, A0249) and β tubulin (Abclonal, U.S.A., 1:5000, AC008) for nuclear and cytoplasmic markers respectively.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Western Blot, Transfection, Knockdown

Journal: Biochemical Journal

Article Title: JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

doi: 10.1042/BCJ20243020

Figure Lengend Snippet: (A) Myc- tagged deletion constructs of YBX1. CoIP of full length JMJD6 with (B) pYCTD, ( C ) pYCSD-CTD, ( D ) pYA/P-CSD. ( E ) Deletion constructs of JMJD6 tagged with V5. CoIP of full length YBX1 with F) pJΔCTD, and G) pJMJC (* denotes endogenous Myc)

Article Snippet: Antibodies used for Western blots were JMJD6 for total protein (endogenous and exogenous) (PSR-H7, Santacruz Biotechnology sc-28348, USA, 1:1000), V5 for exogenously expressed JMJD6-V5 (Thermo Scientific, U.S.A., R960-25, 1:5000), YBX1 (exogenous and endogenous) (Abcam, UK, ab-12148, 1:2000), Myc for exogenously expressed Myc-YBX1 (Sigma Aldrich, U.S.A., 05–419, 1:2000), β-Actin as internal control (Thermo Scientific, U.S.A., A2228, 1:5000), Lamin A/C (Abclonal, U.S.A., 1:5000, A0249) and β tubulin (Abclonal, U.S.A., 1:5000, AC008) for nuclear and cytoplasmic markers respectively.

Techniques: Construct

Journal: Biochemical Journal

Article Title: JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

doi: 10.1042/BCJ20243020

Figure Lengend Snippet: (A) Details of HOTAIR promoter constructs. ( B ) Luciferase assays in MCF7 cells with transient knockout of JMJD6 (JKO) and YBX1(YKO). Dotted line represents luciferase activity in empty vector transfected cells normalized to 1. Extent of protein depletion is shown by western blots in the lower panel. ( C ) Luciferase assay in Vec and JOE cells after YBX1 KO. Vec cells were used as control and in each biological replicate RLA of constructs in Vec cells was normalized to 1. Fold change in activity was calculated over Vec cells.

Article Snippet: Antibodies used for Western blots were JMJD6 for total protein (endogenous and exogenous) (PSR-H7, Santacruz Biotechnology sc-28348, USA, 1:1000), V5 for exogenously expressed JMJD6-V5 (Thermo Scientific, U.S.A., R960-25, 1:5000), YBX1 (exogenous and endogenous) (Abcam, UK, ab-12148, 1:2000), Myc for exogenously expressed Myc-YBX1 (Sigma Aldrich, U.S.A., 05–419, 1:2000), β-Actin as internal control (Thermo Scientific, U.S.A., A2228, 1:5000), Lamin A/C (Abclonal, U.S.A., 1:5000, A0249) and β tubulin (Abclonal, U.S.A., 1:5000, AC008) for nuclear and cytoplasmic markers respectively.

Techniques: Construct, Luciferase, Knock-Out, Activity Assay, Plasmid Preparation, Transfection, Western Blot, Control

Journal: Biochemical Journal

Article Title: JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

doi: 10.1042/BCJ20243020

Figure Lengend Snippet: ( A ) YBX1 ChIP. ( B ) YBX1 ChIP followed by JMJD6 pull down (seqJMJD6) and (C) vice versa. ( D ) JMJD6 ChIP in mock transfected (MT) and YKO cells. YBX1 protein levels are shown by western blot (inset).

Article Snippet: Antibodies used for Western blots were JMJD6 for total protein (endogenous and exogenous) (PSR-H7, Santacruz Biotechnology sc-28348, USA, 1:1000), V5 for exogenously expressed JMJD6-V5 (Thermo Scientific, U.S.A., R960-25, 1:5000), YBX1 (exogenous and endogenous) (Abcam, UK, ab-12148, 1:2000), Myc for exogenously expressed Myc-YBX1 (Sigma Aldrich, U.S.A., 05–419, 1:2000), β-Actin as internal control (Thermo Scientific, U.S.A., A2228, 1:5000), Lamin A/C (Abclonal, U.S.A., 1:5000, A0249) and β tubulin (Abclonal, U.S.A., 1:5000, AC008) for nuclear and cytoplasmic markers respectively.

Techniques: Transfection, Western Blot

Journal: Biochemical Journal

Article Title: JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

doi: 10.1042/BCJ20243020

Figure Lengend Snippet: ( A ) qRT-PCR of HOTAIR in JKO and YKO cells. Dotted line represents HOTAIR levels in control MT cells, normalized to 1. ( B ) qRT-PCR of HOTAIR in MCF7 cells after transient overexpression of the two proteins. Expression levels of empty vector are marked as a dotted line and converted to 1. ( C ) qRT-PCR of HOTAIR in Vec and JOE cells following YBX1 siRNA mediated KD. HOTAIR levels in scrambled siRNA (si_Scr) were used as control and converted to 1. Western blots are shown in lower panel. ( D ) Survival analysis using all three genes (JMJD6, YBX1, HOTAIR), red line denotes high expression of all three and black line denotes low expression of all three genes. Lower panel represents patient number.

Article Snippet: Antibodies used for Western blots were JMJD6 for total protein (endogenous and exogenous) (PSR-H7, Santacruz Biotechnology sc-28348, USA, 1:1000), V5 for exogenously expressed JMJD6-V5 (Thermo Scientific, U.S.A., R960-25, 1:5000), YBX1 (exogenous and endogenous) (Abcam, UK, ab-12148, 1:2000), Myc for exogenously expressed Myc-YBX1 (Sigma Aldrich, U.S.A., 05–419, 1:2000), β-Actin as internal control (Thermo Scientific, U.S.A., A2228, 1:5000), Lamin A/C (Abclonal, U.S.A., 1:5000, A0249) and β tubulin (Abclonal, U.S.A., 1:5000, AC008) for nuclear and cytoplasmic markers respectively.

Techniques: Quantitative RT-PCR, Control, Over Expression, Expressing, Plasmid Preparation, Western Blot