Journal: Nucleic Acids Research

Article Title: Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

doi: 10.1093/nar/gkab258

Figure Lengend Snippet: Toc-HDO-dependent gene silencing of the identified proteins. ( A ) Hepa1–6 cells were used to generate subcell lines stably expressing shRNA against no target (ctrl), luciferase , ANXA5 , CA8 , RNase H1 , APEX1 and FEN1 . Immunoblots performed with the extracts from shRNA knocked down Hepa1–6 cells. ( B ) Toc-Malat1 dose-dependent gene expression regulation analyzed by RT-qPCR ( n = 3). ( C and D ) Quantitative analysis of the substrates uptake of (C) Toc-Malat1 effect ( n = 4), (D) ASO effect ( n = 4). ( E – H ) Quantitative analysis of the (E) Toc-Malat1 effect with 25 nM ( n = 3, light gray), 50 nM ( n = 5, gray), and 100 nM ( n = 4, dark gray), (F) 100 nM of Toc-DMPK effect ( n = 4), (G) 100 nM of Toc-ApoB effect ( n = 5) or (H) 500 nM of ASO effect against Malat1 ( n = 4).

Article Snippet: Antibodies against ACTIN, ANXA2, ANXA5, APEX1, CA8, FEN1, RNase H1, and TPM3 were obtained from Abcam (APEX1 #ab137708 and FEN1 #ab153825 for immunoblots), GeneTex (APEX1 #GTX110558 for immunoprecipitation), ProteinTech (ANXA2 #11256–1-AP and CA8 #12391-1-AP for immunoblots and immunoprecipitation, ANXA5 #11060-1-AP and RNase H1 #15606-1-AP for immunoblots, and TPM3 #10737–1-AP for immunoprecipitation), Cell Signaling Technology (normal Rabbit IgG #3900 (DAE1) for immunoprecipitaion), and WAKO (ACTIN #017-24573 for immunoblot).

Techniques: Stable Transfection, Expressing, shRNA, Luciferase, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

doi: 10.1093/nar/gkab258

Figure Lengend Snippet: In vitro binding assays with purified recombinant proteins. ( A ) CBB stained polyacrylamide gel depicting purified recombinant ANXA5 and CA8 (left panel) and immunoblotting of recombinant CA8 proteins with either an anti-CA8 antibody or anti-His antibody (right panel). ( B and C ) Binding assays of recombinant ANXA5 (0, 250, 500 and 1000 nM) and CA8 (0, 500, 1000 and 2000 nM) with (B) Toc-Malat1 and (C) Toc-DMPK. ( D ) CBB stained polyacrylamide gel depicting purified recombinant APEX1 and FEN1. ( E and F ) In vitro binding assays of Toc-HDO substrates incubated with recombinant APEX1 and FEN1 in (E) N buffer (APEX1: 50, 100 and 150 nM; FEN1: 50, 100 and 150 nM), (F) M buffer with APEX1, FEN1 and RNase H (APEX1: 50, 100 and 150 nM; FEN1: 50, 100 and 150 nM; RNase H: 5, 10 and 20 mU). ( G ) Complete binding of Toc-HDO substrates by high dose of APEX1 and FEN1 in M buffer (APEX1: 100, 200 and 400 nM; FEN1: 100, 200 and 400 nM; RNase H: 1, 2.5, 5 mU). ( H ) Cation-dependent binding pattern of APEX1 (APEX1: 2.7, 27 and 270 nM). ( I ) Substrate length-dependent binding assays with ANXA5, CA8, APEX1 and FEN1 (1000, 1000, 150, 150 nM, respectively). ( J ) In vitro binding assays with or without Toc 13-mer substrates with ANXA5, CA8, APEX1 and FEN1 (1000, 1000, 150, 150 nM, respectively).

Article Snippet: Antibodies against ACTIN, ANXA2, ANXA5, APEX1, CA8, FEN1, RNase H1, and TPM3 were obtained from Abcam (APEX1 #ab137708 and FEN1 #ab153825 for immunoblots), GeneTex (APEX1 #GTX110558 for immunoprecipitation), ProteinTech (ANXA2 #11256–1-AP and CA8 #12391-1-AP for immunoblots and immunoprecipitation, ANXA5 #11060-1-AP and RNase H1 #15606-1-AP for immunoblots, and TPM3 #10737–1-AP for immunoprecipitation), Cell Signaling Technology (normal Rabbit IgG #3900 (DAE1) for immunoprecipitaion), and WAKO (ACTIN #017-24573 for immunoblot).

Techniques: In Vitro, Binding Assay, Purification, Recombinant, Staining, Western Blot, Incubation

Journal: Nucleic Acids Research

Article Title: Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

doi: 10.1093/nar/gkab258

Figure Lengend Snippet: Characterization of identified proteins. ( A ) Fluorescence polarization assays of Toc-HDO substrates incubated with recombinant ANXA5, CA8, APEX1, and FEN1 proteins (10 −9 to 10 −5.5 M). ( B and C ) In vitro binding assays with ANXA5, CA8, APEX1, and FEN1 (1000, 1000, 150 and 150 nM, respectively) and Toc-HDO substrates with (B) PS ASO backbone and (C) PO ASO backbones. ( D ) Pull-down experiments using Toc-biotin–HDO and biotin–ASO of Malat1.

Article Snippet: Antibodies against ACTIN, ANXA2, ANXA5, APEX1, CA8, FEN1, RNase H1, and TPM3 were obtained from Abcam (APEX1 #ab137708 and FEN1 #ab153825 for immunoblots), GeneTex (APEX1 #GTX110558 for immunoprecipitation), ProteinTech (ANXA2 #11256–1-AP and CA8 #12391-1-AP for immunoblots and immunoprecipitation, ANXA5 #11060-1-AP and RNase H1 #15606-1-AP for immunoblots, and TPM3 #10737–1-AP for immunoprecipitation), Cell Signaling Technology (normal Rabbit IgG #3900 (DAE1) for immunoprecipitaion), and WAKO (ACTIN #017-24573 for immunoblot).

Techniques: Fluorescence, Incubation, Recombinant, In Vitro, Binding Assay