NB100-2130  (novus biologicals)

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Antibodies used in this work.

Article Snippet: Ribosomal protein L26 , Novus Biologicals , NB100-2130 , Rabbit , ChIP , https://www.novusbio.com/products/ribosomal-protein-l26-antibody_nb100-2130.

Techniques: Biomarker Discovery

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Primers used in this work.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Sequencing

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Antibodies used in this work.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Biomarker Discovery

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: shRNA lentiviruses obtained from a MISSION ® shRNA Library.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: shRNA, Sequencing

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Demonstration of RPL26, RPL4, RPL8, and RPS9 , as CD40 transcription regulators in human B cells. ( A ) Knockdown of RPL26, RPL4, RPL8 , and RPS9 , in human BL2 cells using shRNA as shown by real time PCR. ( B ) Western blots showing downregulation of RPL26, RPL4, RPL8, and RPS9 results in decreased expression of CD40 , but not CCR6 in BL2 cells. Whole cell protein was used for Western blots. ( C ) Statistical analysis of Western blots in ( B ) based on relative density. NS: not significant. ( D ) Down-regulation of CD40 expression on mRNA levels in the knockdown cells, suggesting a transcriptional regulation of CD40 expression. α-Tubulin was used for loading control. CCR6 was used as a negative control for translational regulation. ( E ) FACS analysis of CD4 0 surface expression in the RPL26, RPL4, RPL8, and RPS9 shRNA knockdown BL2 cells. Scrambled: scrambled shRNA control. sh: shRNA. **: p value < 0.01.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Knockdown, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control, Negative Control

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Demonstration of RPL26, RPL4, RPL8 , and RPS9 as CD40 transcription regulators in human FLS. ( A ) Real time PCR showing downregulation of RPL26 , RPL4 , RPL8 , and RPS9 in FLS by siRNA. ( B ) Western blot showing reduced CD40 expression in RPL26, RPL4, RPL8 , and RPS9 knockdown cells by siRNA. CCR6 was used as a negative control for translational regulation. ( C ) Statistical analysis of Western blots in ( B ) based on relative density. NS: not significant. ( D ) Real time PCR showing reduced CD40 expression in downregulation of RPL26, RPL4, RPL8, and RPS9 in FLS. si: siRNA.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Knockdown, Negative Control

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Demonstration of the binding of RPL26, RPL4, RPL8, and RPS9 to rs6032664. ( A ) Allele-imbalanced binding of RPL26, RPL4, RPL8 and RPS9 to the T (risk allele) and A (non-risk allele) of rs6032664 with the statistical analysis based on the relative density. PARP-1 was used as internal loading control and RSRC2 was used as a positive control. ( B ) Luciferase reporter assays with the risk allele T of rs6032664 demonstrate that RPL26, RPL4, RPL8 , and RPS9 are the transcriptional regulators. si: siRNA. ( C ). ChIP assay showing endogenous binding of RPL26 to rs6032664. (mean ± SD, n = 3 biological replicates, Student t-test with 2 tails). sh: shRNA. NS: not significant.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Binding Assay, Control, Positive Control, Luciferase, shRNA

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Regulation of disease-associated risk genes STAT4, TRAF1, ICAM1 , and CD86 by RPL26 via disease-associated TRN linked by CD40-induced NF-κB signal pathway. ( A ) Western blots showing inactivation of NF-κB p65 by dephosphorylation upon CD40L activation in the RPL26 siRNA knockdown FLS. Statistical analysis based on the relative density is shown. ( B ) real time PCR showing downregulation of four disease-associated risk genes STAT4, TRAF1, ICAM1 , and CD86 as the direct targets of NF-κB p65, ( C ) real time PCR showing downregulation of IL6 and IL1β as the direct targets of NF-κB p65. si: siRNA.

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Western Blot, De-Phosphorylation Assay, Activation Assay, Knockdown, Real-time Polymerase Chain Reaction

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: Disease-associated TRN linked by CD40-induced NF-κB signal pathway. ( A ) scheme showing a disease-associated TRN linked by CD40-induced NF-κB signal pathway. Eight proteins including RPL26, RPL4, RPL8, and RPS9 transcriptionally regulate CD40 expression via binding to fSNP rs6032664. Downstream of CD40-induced NF-κB signaling, STAT4, TRAF1, ICAM1 , and CD86 as well as IL6 and IL1β were regulated by NF-κB p65 transcriptionally. FAM76B is a risk gene for MS. ( B ) Scratch-wound assay showing defects in cell migration in RPL26, RPL4, RPL8 , and RPS9 siRNA knockdown FLS. si: siRNA. ( C ) Scratch-wound assay showing defects in cell migration in FLS with CD40 and ICAM1 siRNA knockdown. Downregulation of CD40 and ICAM1 was demonstrated by Western blots as shown in .

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Expressing, Binding Assay, Scratch Wound Assay Assay, Migration, Knockdown, Western Blot

Journal: Genes

Article Title: Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus

doi: 10.3390/genes11121526

Figure Lengend Snippet: ( A ) Microscopy (upper) and DAPI staining (lower) showing no obvious difference on cell morphology between the scrambled shRNA BL2 cells and BL2 cells with shRNA knockdown of RPL26, RPL4, RPL8 and RPS9 . ( B ) Microscopy showing no obvious difference on cell morphology between the scrambled shRNA FLS and FLS with siRNA knockdown of RPL26, RPL4, RPL8 and RPS9 .

Article Snippet: Briefly, scrambled shRNA BL2 cells and RPL26 shRNA knockdown BL2 cells were cross-linked with 1% formaldehyde for 10 min. Sonication was carried out at 30% amplitude with 20s on and 50s off for 5 min. 10 μg anti-ribosomal protein L26 antibody (Novus Biologicals, Littleton, CO, USA)(Cat#: NB100-2130) coupled to DynabeadsTM Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA)(Cat#:10001D and 10003D) was used for immunoprecipitation.

Techniques: Microscopy, Staining, shRNA, Knockdown