anti pum1  (Proteintech)

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: The poly(A) tail is necessary for PUM-mediated repression. ( A ) PUM1&2 repression activity was measured in HCT116 cells using Nano-luciferase reporters with three PREs within a minimal 3′ UTR with cleavage and poly-adenylation signals (Nluc 3×PRE), calculated relative to a version wherein the PRE sequences were mutated (indicated in red text) to prevent PUM binding (Nluc 3×PREmt). PUM repression of the poly-adenylated reporter was compared to a Nluc reporter that has a 3′ end generated by the MALAT1 non-coding RNA (Nluc 3×PRE MALAT), which is processed by RNase P mediated cleavage to form a triple helix structure (PBD: 4PLX). Derivatives of the Nluc 3×PRE were constructed with internal poly(A) tracts of either 20 (A20) or 60 (A60) adenosines, inserted between the PREs and the MALAT1 triple helix. Firefly luciferase (Fluc) served as an internal control to normalize transfection efficiency. ( B ) PRE dependent repression by endogenous PUM1 and PUM2 was measured as log(2) fold change of each Nluc 3×PRE reporter relative to its corresponding 3×PREmt reporter. Mean fold change is plotted along with individual replicate data points. n = 9; three experiments, each with three biological replicates; ± standard deviation (SD). For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Asterisks above the axis denote significance relative to the 3×PREmt version of each reporter type, whereas below the bars are calculated relative to the poly-adenylated Nluc reporters. ( C ) Western blot confirming the depletion of PAN2 and CNOT1 proteins by RNAi in HCT116 cells. GAPDH served as a loading control. n = 3 experimental replicates. ( D ) The effect of CCR4-NOT or PAN2 and PAN3 knockdown on PUM repression of the Nluc 3×PRE reporter, relative to the mutant Nluc 3×PREmt, was measured in HCT116 cells, in comparison to cells transfected with NTC siRNAs. n = 9; three experiments, each with three biological replicates; ± SD.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed three times with 250 μl 1×PBS + 0.1% Tween-20 and incubated in 100 μl of 1×PBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl Labs, A302-576A) overnight, rotating at 4°C.

Techniques: Activity Assay, Luciferase, Binding Assay, Generated, Construct, Control, Transfection, Standard Deviation, Western Blot, Knockdown, Mutagenesis, Comparison

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUM1 interacts with endogenous PABPCs through the RRM domain independent of RNA. ( A ) PABPC1 and PABPC4 co-immunoprecipitate with PUM1 from HCT116 cell extracts treated with RNases A and One. PUM1, eIF4E, eIF4G, PABPC1, PABPC4, and GAPDH were detected by western blot. ( B ) Denaturing formaldehyde agarose gel analysis confirmed depletion of RNA in the HCT116 cell extracts before (−) or after treatment with RNase A and RNase One (+). The 18S and 28S rRNA bands, detected by ethidium bromide, are indicated on the right. ( C ) Human PABPC1 domain architecture showing the N-terminal RRM domains with the critical eIF4G binding site residues and the proline-rich linker and C-terminal region PABC domain containing the MLLE motif residues important binding by PAM2-domain containing proteins. ( D ) Western blot of the co-immunoprecipitation analysis of endogenous PUM1 with either HaloTag (HT), as a negative control, or HT-PABPC1 full-length (aa1–636) or HT-RRMs 1–4 (aa 1–370). All input samples were treated with RNase A and One. IgG beads and GAPDH served as negative controls. Dashed vertical lines in the panels indicate that the images were cropped to show relevant lanes. ( E ) Western blot of PUM1 immunoprecipitates to detect association with either wild-type full-length HT-PABPC1, or mutant versions wherein the eIF4G binding site is mutated (M161A and D165A) or the MLLE motif is mutated to (MLLEmt: M584G, L585A, L586A, and E587R), or the HT-MLLE domain (aa 542–636). All input samples were treated with RNase A and One. ( F ) Western blot of the co-immunoprecipitation analysis of endogenous PUM1 to detect association with either wild-type full-length HT-PABPC1 or the RNA-binding mutant version (RBmt). All input samples were treated with RNase A and One. HT and H3 served as negative controls.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed three times with 250 μl 1×PBS + 0.1% Tween-20 and incubated in 100 μl of 1×PBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl Labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Agarose Gel Electrophoresis, Binding Assay, Immunoprecipitation, Negative Control, Mutagenesis, RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: Poly(A)-binding proteins PABPC1 and PABPC4 are necessary for PUM-mediated repression. ( A ) Western blot analysis confirms auxin induced depletion of degron-tagged PABPC1-AID and RNAi depletion of PABPC4 either individually or in combination in HCT116 cells. RNAi was performed by transfecting cells with either PABPC4 or NTC siRNAs. PABPC1-AID protein was depleted upon treatment with indole-3-acetic acid (+IAA), in comparison to vehicle only control (−IAA). GAPDH served as a loading control. ( B )PUM-mediated repression of Nluc 3×PRE reporter expression was measured in HCT116 cells, relative to the mutant PRE version. Individual and combined effects of PABPC4 RNAi and IAA-induced depletion of PABPC1-AID on PUM repression were tested. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, **** P < .001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( C ) Western blot analysis of samples from a representative experimental replicate for experiments shown in panels ( D ) and ( E ). GAPDH served as a loading control and PUM1 and PUM2 protein levels were detected as controls. The effect of combined PABPC4 RNAi and PABPC1-AID depletion on PUM repression of Nluc reporters containing the 3′ UTRs of the natural, PRE-containing, PUM target mRNAs FZD8 in panel (D) and CDKN1B in panel (E) were measured, relative to matched PRE mutant versions. n = 9; three experiments, each with three biological replicates; ± SD. ( F ) Live cell numbers were measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD. ( G ) Cell viability was measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed three times with 250 μl 1×PBS + 0.1% Tween-20 and incubated in 100 μl of 1×PBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl Labs, A302-576A) overnight, rotating at 4°C.

Techniques: Binding Assay, Western Blot, Comparison, Control, Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUMs and PABPCs have opposing effects on mRNA stability. ( A ) Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RT-qPCR in RNA samples purified from HCT116 cells wherein PUM1 and PUM2 were depleted by RNAi. Changes in mRNA levels were determined relative to cells treated with the NTC siRNAs. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. ( B ) Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RT-qPCR in RNA samples purified from HCT116 cells wherein PABPC1-AID and PABPC4 were depleted by auxin treatment (+IAA) and RNAi, respectively. Changes in mRNA levels were determined relative to cells treated with vehicle only (−IAA) and the NTC siRNA. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. In both panels (A) and (B), transcript level was measured by RT-qPCR, normalized to 18S rRNA, and plotted as fold change relative to the non-depleted control condition. n = 3 biological replicates, ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, ns = not significant based on unpaired two-tailed t -tests. ( C ) Western blot analysis confirmed RNAi depletion of PUM1 and PUM2 in biological replicates in panel A. GAPDH served as a loading control. ( D ) Western blot analysis of PABPC1-AID and PABPC4 confirmed their depletion by auxin induced degradation and RNAi, respectively, in samples from panel (B). Histone H3 served as a loading control.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed three times with 250 μl 1×PBS + 0.1% Tween-20 and incubated in 100 μl of 1×PBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl Labs, A302-576A) overnight, rotating at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Two Tailed Test, Western Blot

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC overexpression alleviates PUM repression and requires RNA binding. ( A ) Western blot analysis of halotag (HT) and HT-PABPC1 titration for samples from a representative experimental replicate of panel (B). The amount of transfected plasmid for each effector is shown at the top. GAPDH served as a loading control. PUM1 and PUM2 were detected as an additional control. ( B ) Reporter assay showing the effect of HT-PABPC1 overexpression on PUM repression of the Nluc 3×PRE reporter in wild-type HCT116 cells, calculated relative to the Nluc 3×PREmt reporter. Halotag served as a negative control. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and *** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Significance indicated above the X-axis indicates relative to 3×PREmt reporter, whereas significance calling shown below is indicated by the respective brackets. ( C ) Graph of the fold change in HT-PABPC1 exogenous expression over endogenous PABPC1 levels calculated from three experimental replicates including the western blot shown in panel (A). n = 3; ± SD. ( D ) Western blot analysis of halotag (HT), HT-PABPC1 full-length, HT-PABPC1 full-length RNA-binding mutant (RBmt), HT-RRM1-4 domains, and HT-RRM1-4 RBmt samples taken from a representative experimental replicate of panel (E). GAPDH served as a loading control. PUM1 and PUM2 were detected as controls. ( E ) Reporter assay showing effect of HT-PABPC1 full-length, HT-RRM1-4 domains, and RNA-binding mutants versions when overexpressed on PUM repression of the Nluc 3×PRE reporter in HCT116 cells. n = 9; three experiments, each with three biological replicates; ± SD. Halotag served as a negative control.( F ) Western blot analysis of overexpressed HT-PABPC1 on PUM repression and the effect of the mutation of the eIF4G binding site mutant (M161A, D165A), or the MLLE motif (MLLEmt), or the RNA-binding defective mutant (RBmt) from a representative experimental replicate of panel (G). H3 served as a loading control. PUM1 and PUM2 were detected as additional controls. ( G ) Reporter assay showing effect of HT-PABPC1 full-length mutants on PUM repression in wild-type HCT116 cells. n = 9; three experiments, each with three biological replicates; ± SD. Halotag served as a negative control.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed three times with 250 μl 1×PBS + 0.1% Tween-20 and incubated in 100 μl of 1×PBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl Labs, A302-576A) overnight, rotating at 4°C.

Techniques: Over Expression, RNA Binding Assay, Western Blot, Titration, Transfection, Plasmid Preparation, Control, Reporter Assay, Negative Control, Expressing, Mutagenesis, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC1 does not disrupt PUM binding to Nluc 3×PRE mRNA. ( A ) Co-immunoprecipitation analysis of PUM1 binding to the Nluc 3×PRE reporter in the presence of overexpressed halotag (HT) or HT-PABPC1. The top two panels show western blot detection of PUM1 protein and HT-PABPC1 in the 2% of the input HCT116 cell extracts and 25% of the PUM1 RNA co-immunoprecipitates (RIP) from three biological replicate samples per condition. The bottom panel shows detection of Nluc mRNA by northern blotting. For northern blots, 1% of input samples and 75% of the RIP samples were loaded on the formaldehyde-MOPS agarose gel, respectively. Co-immunoprecipitation with IgG beads served as a negative control. ( B ) Fold increase of the Nluc 3×PRE mRNA in the input samples from cells expressing HT-PABPC1 are plotted relative to the HT control, based on data in panel (A). n = 3 biological replicates, ± SD. For significance calling, *** P < .001 based on an unpaired student’s t -test. ( C ) Fold enrichment of the Nluc 3×PRE mRNA in PUM1 RIP samples was measured relative to the IgG control. Importantly, the mRNA levels in each RIP sample was normalized to that present in the respective input samples. n = 3 biological replicates, ± SD. For significance calling, the difference between HT and HT-PABPC1 was not significant ( P > 0.05 = ns) based on an unpaired student’s t -test.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed three times with 250 μl 1×PBS + 0.1% Tween-20 and incubated in 100 μl of 1×PBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl Labs, A302-576A) overnight, rotating at 4°C.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Negative Control, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: The poly(A) tail is necessary for PUM-mediated repression. ( A ) PUM1&2 repression activity was measured in HCT116 cells using Nano-luciferase reporters with three PREs within a minimal 3′ UTR with cleavage and poly-adenylation signals (Nluc 3×PRE), calculated relative to a version wherein the PRE sequences were mutated (indicated in red text) to prevent PUM binding (Nluc 3×PREmt). PUM repression of the poly-adenylated reporter was compared to a Nluc reporter that has a 3′ end generated by the MALAT1 non-coding RNA (Nluc 3×PRE MALAT), which is processed by RNase P mediated cleavage to form a triple helix structure (PBD: 4PLX). Derivatives of the Nluc 3×PRE were constructed with internal poly(A) tracts of either 20 (A20) or 60 (A60) adenosines, inserted between the PREs and the MALAT1 triple helix. Firefly luciferase (Fluc) served as an internal control to normalize transfection efficiency. ( B ) PRE dependent repression by endogenous PUM1 and PUM2 was measured as log(2) fold change of each Nluc 3×PRE reporter relative to its corresponding 3×PREmt reporter. Mean fold change is plotted along with individual replicate data points. n = 9; three experiments, each with three biological replicates; ± standard deviation (SD). For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Asterisks above the axis denote significance relative to the 3×PREmt version of each reporter type, whereas below the bars are calculated relative to the poly-adenylated Nluc reporters. ( C ) Western blot confirming the depletion of PAN2 and CNOT1 proteins by RNAi in HCT116 cells. GAPDH served as a loading control. n = 3 experimental replicates. ( D ) The effect of CCR4-NOT or PAN2 and PAN3 knockdown on PUM repression of the Nluc 3×PRE reporter, relative to the mutant Nluc 3×PREmt, was measured in HCT116 cells, in comparison to cells transfected with NTC siRNAs. n = 9; three experiments, each with three biological replicates; ± SD.

Article Snippet: For each sample, 20 μl of magnetic protein A surebeads (Bio-Rad) were equilibrated three times 250 μl 1× phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific) + 0.1% Tween-20 and incubated in 100 μl 1× PBS + 0.1% Tween-20 + 2 μg PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Activity Assay, Luciferase, Binding Assay, Generated, Construct, Control, Transfection, Standard Deviation, Western Blot, Knockdown, Mutagenesis, Comparison

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUM1 interacts with endogenous PABPCs through the RRM domain independent of RNA. ( A ) PABPC1 and PABPC4 co-immunoprecipitate with PUM1 from HCT116 cell extracts treated with RNases A and One. PUM1, eIF4E, eIF4G, PABPC1, PABPC4, and GAPDH were detected by western blot. ( B ) Denaturing formaldehyde agarose gel analysis confirmed depletion of RNA in the HCT116 cell extracts before (−) or after treatment with RNase A and RNase One (+). The 18S and 28S rRNA bands, detected by ethidium bromide, are indicated on the right. ( C ) Human PABPC1 domain architecture showing the N-terminal RRM domains with the critical eIF4G binding site residues and the proline-rich linker and C-terminal region PABC domain containing the MLLE motif residues important binding by PAM2-domain containing proteins. ( D ) Western blot of the co-immunoprecipitation analysis of endogenous PUM1 with either HaloTag (HT), as a negative control, or HT-PABPC1 full-length (aa1–636) or HT-RRMs 1–4 (aa 1–370). All input samples were treated with RNase A and One. IgG beads and GAPDH served as negative controls. Dashed vertical lines in the panels indicate that the images were cropped to show relevant lanes. ( E ) Western blot of PUM1 immunoprecipitates to detect association with either wild-type full-length HT-PABPC1, or mutant versions wherein the eIF4G binding site is mutated (M161A and D165A) or the MLLE motif is mutated to (MLLEmt: M584G, L585A, L586A, and E587R), or the HT-MLLE domain (aa 542–636). All input samples were treated with RNase A and One. ( F ) Western blot of the co-immunoprecipitation analysis of endogenous PUM1 to detect association with either wild-type full-length HT-PABPC1 or the RNA-binding mutant version (RBmt). All input samples were treated with RNase A and One. HT and H3 served as negative controls.

Article Snippet: For each sample, 20 μl of magnetic protein A surebeads (Bio-Rad) were equilibrated three times 250 μl 1× phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific) + 0.1% Tween-20 and incubated in 100 μl 1× PBS + 0.1% Tween-20 + 2 μg PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Agarose Gel Electrophoresis, Binding Assay, Immunoprecipitation, Negative Control, Mutagenesis, RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: Poly(A)-binding proteins PABPC1 and PABPC4 are necessary for PUM-mediated repression. ( A ) Western blot analysis confirms auxin induced depletion of degron-tagged PABPC1-AID and RNAi depletion of PABPC4 either individually or in combination in HCT116 cells. RNAi was performed by transfecting cells with either PABPC4 or NTC siRNAs. PABPC1-AID protein was depleted upon treatment with indole-3-acetic acid (+IAA), in comparison to vehicle only control (−IAA). GAPDH served as a loading control. ( B )PUM-mediated repression of Nluc 3×PRE reporter expression was measured in HCT116 cells, relative to the mutant PRE version. Individual and combined effects of PABPC4 RNAi and IAA-induced depletion of PABPC1-AID on PUM repression were tested. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, **** P < .001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( C ) Western blot analysis of samples from a representative experimental replicate for experiments shown in panels ( D ) and ( E ). GAPDH served as a loading control and PUM1 and PUM2 protein levels were detected as controls. The effect of combined PABPC4 RNAi and PABPC1-AID depletion on PUM repression of Nluc reporters containing the 3′ UTRs of the natural, PRE-containing, PUM target mRNAs FZD8 in panel (D) and CDKN1B in panel (E) were measured, relative to matched PRE mutant versions. n = 9; three experiments, each with three biological replicates; ± SD. ( F ) Live cell numbers were measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD. ( G ) Cell viability was measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD.

Article Snippet: For each sample, 20 μl of magnetic protein A surebeads (Bio-Rad) were equilibrated three times 250 μl 1× phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific) + 0.1% Tween-20 and incubated in 100 μl 1× PBS + 0.1% Tween-20 + 2 μg PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Binding Assay, Western Blot, Comparison, Control, Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUMs and PABPCs have opposing effects on mRNA stability. ( A ) Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RT-qPCR in RNA samples purified from HCT116 cells wherein PUM1 and PUM2 were depleted by RNAi. Changes in mRNA levels were determined relative to cells treated with the NTC siRNAs. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. ( B ) Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RT-qPCR in RNA samples purified from HCT116 cells wherein PABPC1-AID and PABPC4 were depleted by auxin treatment (+IAA) and RNAi, respectively. Changes in mRNA levels were determined relative to cells treated with vehicle only (−IAA) and the NTC siRNA. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. In both panels (A) and (B), transcript level was measured by RT-qPCR, normalized to 18S rRNA, and plotted as fold change relative to the non-depleted control condition. n = 3 biological replicates, ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, ns = not significant based on unpaired two-tailed t -tests. ( C ) Western blot analysis confirmed RNAi depletion of PUM1 and PUM2 in biological replicates in panel A. GAPDH served as a loading control. ( D ) Western blot analysis of PABPC1-AID and PABPC4 confirmed their depletion by auxin induced degradation and RNAi, respectively, in samples from panel (B). Histone H3 served as a loading control.

Article Snippet: For each sample, 20 μl of magnetic protein A surebeads (Bio-Rad) were equilibrated three times 250 μl 1× phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific) + 0.1% Tween-20 and incubated in 100 μl 1× PBS + 0.1% Tween-20 + 2 μg PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Two Tailed Test, Western Blot

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC overexpression alleviates PUM repression and requires RNA binding. ( A ) Western blot analysis of halotag (HT) and HT-PABPC1 titration for samples from a representative experimental replicate of panel (B). The amount of transfected plasmid for each effector is shown at the top. GAPDH served as a loading control. PUM1 and PUM2 were detected as an additional control. ( B ) Reporter assay showing the effect of HT-PABPC1 overexpression on PUM repression of the Nluc 3×PRE reporter in wild-type HCT116 cells, calculated relative to the Nluc 3×PREmt reporter. Halotag served as a negative control. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and *** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Significance indicated above the X-axis indicates relative to 3×PREmt reporter, whereas significance calling shown below is indicated by the respective brackets. ( C ) Graph of the fold change in HT-PABPC1 exogenous expression over endogenous PABPC1 levels calculated from three experimental replicates including the western blot shown in panel (A). n = 3; ± SD. ( D ) Western blot analysis of halotag (HT), HT-PABPC1 full-length, HT-PABPC1 full-length RNA-binding mutant (RBmt), HT-RRM1-4 domains, and HT-RRM1-4 RBmt samples taken from a representative experimental replicate of panel (E). GAPDH served as a loading control. PUM1 and PUM2 were detected as controls. ( E ) Reporter assay showing effect of HT-PABPC1 full-length, HT-RRM1-4 domains, and RNA-binding mutants versions when overexpressed on PUM repression of the Nluc 3×PRE reporter in HCT116 cells. n = 9; three experiments, each with three biological replicates; ± SD. Halotag served as a negative control.( F ) Western blot analysis of overexpressed HT-PABPC1 on PUM repression and the effect of the mutation of the eIF4G binding site mutant (M161A, D165A), or the MLLE motif (MLLEmt), or the RNA-binding defective mutant (RBmt) from a representative experimental replicate of panel (G). H3 served as a loading control. PUM1 and PUM2 were detected as additional controls. ( G ) Reporter assay showing effect of HT-PABPC1 full-length mutants on PUM repression in wild-type HCT116 cells. n = 9; three experiments, each with three biological replicates; ± SD. Halotag served as a negative control.

Article Snippet: For each sample, 20 μl of magnetic protein A surebeads (Bio-Rad) were equilibrated three times 250 μl 1× phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific) + 0.1% Tween-20 and incubated in 100 μl 1× PBS + 0.1% Tween-20 + 2 μg PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Over Expression, RNA Binding Assay, Western Blot, Titration, Transfection, Plasmid Preparation, Control, Reporter Assay, Negative Control, Expressing, Mutagenesis, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC1 does not disrupt PUM binding to Nluc 3×PRE mRNA. ( A ) Co-immunoprecipitation analysis of PUM1 binding to the Nluc 3×PRE reporter in the presence of overexpressed halotag (HT) or HT-PABPC1. The top two panels show western blot detection of PUM1 protein and HT-PABPC1 in the 2% of the input HCT116 cell extracts and 25% of the PUM1 RNA co-immunoprecipitates (RIP) from three biological replicate samples per condition. The bottom panel shows detection of Nluc mRNA by northern blotting. For northern blots, 1% of input samples and 75% of the RIP samples were loaded on the formaldehyde-MOPS agarose gel, respectively. Co-immunoprecipitation with IgG beads served as a negative control. ( B ) Fold increase of the Nluc 3×PRE mRNA in the input samples from cells expressing HT-PABPC1 are plotted relative to the HT control, based on data in panel (A). n = 3 biological replicates, ± SD. For significance calling, *** P < .001 based on an unpaired student’s t -test. ( C ) Fold enrichment of the Nluc 3×PRE mRNA in PUM1 RIP samples was measured relative to the IgG control. Importantly, the mRNA levels in each RIP sample was normalized to that present in the respective input samples. n = 3 biological replicates, ± SD. For significance calling, the difference between HT and HT-PABPC1 was not significant ( P > 0.05 = ns) based on an unpaired student’s t -test.

Article Snippet: For each sample, 20 μl of magnetic protein A surebeads (Bio-Rad) were equilibrated three times 250 μl 1× phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific) + 0.1% Tween-20 and incubated in 100 μl 1× PBS + 0.1% Tween-20 + 2 μg PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Negative Control, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Impact of Low-Dose CT Radiation on Gene Expression and DNA Integrity

doi: 10.3390/ijms262411869

Figure Lengend Snippet: Jitter plots illustrate the distribution of differential gene expression (DGE) after CT scan for the indicated genes in the analysis of all samples (in vivo and ex vivo combined). The graphs show DGE normalized with PUM1 and relative to the unexposed in vivo blood sample. Black dots represent the median, with the whiskers representing the interquartile range (25th–75th percentiles) and the dotted line the baseline gene expression. Statistical analysis was performed in log2 transformed data. A single asterisk indicates a gene that is significantly differentially expressed compared with the control at a significance level of p < 0.05, and two asterisks denote a significance level of p < 0.001.

Article Snippet: Equal quantities of cDNA were mixed with TaqManTM Universal PCR Master Mix (Applied BiosystemsTM, Thermo Fisher Scientific, Waltham, MA, USA) and gene-specific TaqManTM (Thermo Fisher Scientific, Waltham, MA, USA) assays for PUM1 (Hs00206469_m1), AEN (Hs00224322_m1), BAX (Hs00180269_m1), DDB2 (Hs00172068_m1), FDXR (Hs01031617_m1), EDA2R (Hs00939736_m1), MIR34AHG (Hs04333908_s1), PHLDA3 (Hs00385313_m1), POU2AF1 (Hs01573371_m1), and WNT3 (Hs00902257_m1). qRT-PCR was performed using the QuantStudioTM 12K OA Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA), where each gene was measured in duplicate.

Techniques: Gene Expression, Computed Tomography, In Vivo, Ex Vivo, Transformation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Impact of Low-Dose CT Radiation on Gene Expression and DNA Integrity

doi: 10.3390/ijms262411869

Figure Lengend Snippet: Jitter plots illustrate the distribution of differential gene expression (DGE) after CT scan for the indicated genes, with comparison between DGE of in vivo- and ex vivo-incubated samples. The graphs show DGE normalized with PUM1 and relative to the unexposed in vivo blood sample. Black dots represent the median value, with the whiskers representing the interquartile range (25th–75th percentiles) and the dotted line the baseline gene expression. Statistical analysis was performed in log2 transformed data. A single asterisk indicates a gene that is significantly differentially expressed compared with the control at a significance level of p < 0.05, and two asterisks denote a significance level of p < 0.001. Significance levels are solely displayed for the in vivo-incubated samples.

Article Snippet: Equal quantities of cDNA were mixed with TaqManTM Universal PCR Master Mix (Applied BiosystemsTM, Thermo Fisher Scientific, Waltham, MA, USA) and gene-specific TaqManTM (Thermo Fisher Scientific, Waltham, MA, USA) assays for PUM1 (Hs00206469_m1), AEN (Hs00224322_m1), BAX (Hs00180269_m1), DDB2 (Hs00172068_m1), FDXR (Hs01031617_m1), EDA2R (Hs00939736_m1), MIR34AHG (Hs04333908_s1), PHLDA3 (Hs00385313_m1), POU2AF1 (Hs01573371_m1), and WNT3 (Hs00902257_m1). qRT-PCR was performed using the QuantStudioTM 12K OA Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA), where each gene was measured in duplicate.

Techniques: Gene Expression, Computed Tomography, Comparison, In Vivo, Ex Vivo, Incubation, Transformation Assay, Control

Journal: bioRxiv

Article Title: Viral non-coding RNAs hijack host Pumilio proteins to regulate host transcripts

doi: 10.1101/2025.11.27.691032

Figure Lengend Snippet: (A) HSUR1 and HSUR2 contain conserved PREs. Alignment of HSUR1 and HSUR2 indicating binding sites for miRNAs and RNA-binding proteins. Arches indicate base pairing. ARE, AU-rich element; Sm, binding site for Sm proteins; PRE, Pumilio response element. (B) The 3′-terminal stem–loop of HSUR1 and HSUR2 is structurally conserved across multiple HVS isolates and related herpesvirus ateles. Common secondary structure was predicted by RNAalifold . Circles indicate consistent and covarying nucleotides. Red indicates base pairs with no sequence variation; ochre and green indicate base pairs with two and three different types of base pairs, respectively. Mutations in the PRE used in this study are indicated. (C) PUM1 and PUM2 bind both HSUR1 and HSUR2. RNA immunoprecipitation from marmoset T cells transformed with WT HVS using anti-PUM1 antibody, anti-PUM2 antibody, or IgG control. I, input (5%). Input from cells transduced with virus lacking HSUR1 and HSUR2 (Δ2A) was included as an additional negative control. (D) PUM1 and PUM2 levels are not affected by the presence of HSUR1 and HSUR2. Western blot analysis of protein levels in marmoset cells transformed with either WT or Δ2A HVS. (E) PRE mutations do not affect HSUR expression. Representative Northern blot of RNAs from Jurkat cells transduced with the indicated HSUR variants or EV, probed for ncRNAs. (F) HSURs bind PUM proteins via PREs. RNA immunoprecipitation from Jurkat cells transduced with the indicated HSUR variants using anti-PUM1 antibody, anti-PUM2 antibody, or IgG control. I, input (5%).

Article Snippet: The lysate was divided into 3 parts and each was incubated with 30 μl Dynabeads Protein G (Invitrogen) coupled to 10 μg of antibodies: anti-PUM1 (Bethyl), anti-PUM2 (Bethyl), and an IgG control (Sigma).

Techniques: Binding Assay, RNA Binding Assay, Sequencing, RNA Immunoprecipitation, Transformation Assay, Control, Transduction, Virus, Negative Control, Western Blot, Expressing, Northern Blot

Journal: bioRxiv

Article Title: Viral non-coding RNAs hijack host Pumilio proteins to regulate host transcripts

doi: 10.1101/2025.11.27.691032

Figure Lengend Snippet: (A) Only a subset of genes upregulated by HSURs or PUM loss are direct PUM targets. RIP in Jurkat cells using anti-PUM1 (left), anti-PUM2 (right), or IgG control, shown as enrichment over input; mean of 3 independent experiments ± SD. (B) Genes upregulated by HSURs are also induced upon PUM depletion. RNA levels of selected transcripts in polyclonal PUM1 (left) or PUM2 (right) knockouts in Jurkat cells; mean of 3 independent experiments ± SD; *p ≤ 0.05, ***p ≤ 0.001 (paired t-test). (C) Schematic of HSUR2 mutants designed to identify additional elements involved in regulation of PUM-dependent targets. (D) Northern blot showing expression of HSUR2 variants in Jurkat cells. (E) Neither PRE location nor miR-142-3p binding affects HSUR2’s ability to increase PUM-regulated transcripts. Relative mRNA levels of selected transcripts in Jurkat cells expressing the indicated HSUR2 variants; mean of 3 independent experiments ± SD.

Article Snippet: The lysate was divided into 3 parts and each was incubated with 30 μl Dynabeads Protein G (Invitrogen) coupled to 10 μg of antibodies: anti-PUM1 (Bethyl), anti-PUM2 (Bethyl), and an IgG control (Sigma).

Techniques: Control, Northern Blot, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Viral non-coding RNAs hijack host Pumilio proteins to regulate host transcripts

doi: 10.1101/2025.11.27.691032

Figure Lengend Snippet: (A ) Estimated numbers of PUM1 and PUM2 protein molecules per Jurkat cell relative to published values for HCT116 cells. (B) RNA immunoprecipitation in Jurkat cells using anti-PUM1 (left), anti-PUM2 (right), or IgG control, shown as enrichment over input; mean of 3 independent experiments ± SD. (C) Less abundant PUM targets are more strongly regulated by HSURs in a PRE-dependent manner; PUM targets were binned by expression level and PRE-dependent changes in expression are shown for each bin. (D) Schematic of HSUR2 mutants designed to identify additional elements involved in regulation of PUM-dependent targets.

Article Snippet: The lysate was divided into 3 parts and each was incubated with 30 μl Dynabeads Protein G (Invitrogen) coupled to 10 μg of antibodies: anti-PUM1 (Bethyl), anti-PUM2 (Bethyl), and an IgG control (Sigma).

Techniques: RNA Immunoprecipitation, Control, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Prolyl endopeptidase is a multifunctional host factor required for FMDV infection

doi: 10.1007/s00018-025-05941-0

Figure Lengend Snippet: PREP -KO IBRS-2 cells upregulate RIG-I mRNA expression by decreasing PUM1 stability. A Western blot analysis of the expression levels of key molecules involved in the type I IFN signaling pathway in PREP -KO and sgNC IBRS-2 cells. B Western blot analysis of RIG-I protein expression levels in GFP-PREP overexpression and WT IBRS-2 cells. C RT-qPCR analysis of relative RIG-I , MDA5 and MAVS mRNA expression levels in PREP -KO and sgNC IBRS-2 cells. D RT-qPCR analysis of relative PUM1 mRNA expression levels in PREP -KO and sgNC IBRS-2 cells. E Western blot analysis of PUM1 protein expression levels in PREP -KO and sgNC IBRS-2 cells. F RT-qPCR analysis of the relative mRNA expression levels of IFN-β , RIG-I , MDA5 and OASL in IBRS-2 cells transfected with either control siRNA (siNC) or PUM1-targeting siRNA (siPUM1). G Co-immunoprecipitation analysis of lysates from HEK293T cells co-transfected with PREP-Myc and PUM1-Flag. H Western blot analysis of PUM1 expression levels in PREP -KO and sgNC IBRS-2 cells treated with CHX (100 µg/mL) for the indicated times. I Western blot analysis of PUM1 expression levels in PREP -KO and sgNC IBRS-2 cells maintained in the presence of DMSO, MG132 (4 or 20 µM), CQ (20 or 50 µM) or Z-VAD-FMK (10 or 50 µM). J Western blot analysis of PUM1 expression levels in PREP -KO and sgNC IBRS-2 cells maintained in the presence of DMSO, Z-DEVD-FMK (15 µM), Z-IETD-FMK (15 µM) or Z-LEHD-FMK (15 µM). K Dual-luciferase reporter analysis of relative porcine RIG-I promoter activity in IBRS-2 cells transfected with siPUM1 or siNC, followed by transfection with porcine RIG-I dual-luciferase reporter plasmids. sgNC, sgRNA negative control; KO, knock out; WT, wild-type; kDa, kilodaltons. Data are represented as means ± SD. n = 3 ( C , D , F and K ). * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, no significant. P values were determined by two-tailed Student′s t -tests

Article Snippet: Membranes were blocked with 5% skim milk for 1 h and then probed with primary antibodies: PREP (Bethyl, #A305-348 A, 1:1,000), β-actin (Proteintech, #66009-lg, 1:2,000), PUM1 (Proteintech, #26256-1-AP, 1:1,000), VP1 (prepared in our laboratory, 1:1,000), 3D (prepared in our laboratory, 1:1,000), RIG-I (prepared in our laboratory, 1:1,000), TBK1 (Cell Signaling Technology, #3504, 1:1,000), IRF3 (Cell Signaling Technology, #4302, 1:1,000), IκBα (Cell Signaling Technology, #4812, 1:1,000), p65 (Cell Signaling Technology, #4764, 1:1,000), Myc (Cell Signaling Technology, #2278, 1:1,000), Flag (Cell Signaling Technology, #2368, 1:1,000).

Techniques: Expressing, Western Blot, Over Expression, Quantitative RT-PCR, Transfection, Control, Immunoprecipitation, Luciferase, Activity Assay, Negative Control, Knock-Out, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Prolyl endopeptidase is a multifunctional host factor required for FMDV infection

doi: 10.1007/s00018-025-05941-0

Figure Lengend Snippet: PREP is a multifunctional host factor required for FMDV infection. In IBRS-2 cells, PREP interacts with PUM1, while PREP -KO reduces the stability of PUM1. The destabilization of PUM1 enhances porcine RIG-I promoter activity, upregulating RIG-I and MDA5, thereby activating the type I IFN signaling pathway. In other FMDV-susceptible cells, PREP -KO inhibits the FMDV life cycle at the replication stage

Article Snippet: Membranes were blocked with 5% skim milk for 1 h and then probed with primary antibodies: PREP (Bethyl, #A305-348 A, 1:1,000), β-actin (Proteintech, #66009-lg, 1:2,000), PUM1 (Proteintech, #26256-1-AP, 1:1,000), VP1 (prepared in our laboratory, 1:1,000), 3D (prepared in our laboratory, 1:1,000), RIG-I (prepared in our laboratory, 1:1,000), TBK1 (Cell Signaling Technology, #3504, 1:1,000), IRF3 (Cell Signaling Technology, #4302, 1:1,000), IκBα (Cell Signaling Technology, #4812, 1:1,000), p65 (Cell Signaling Technology, #4764, 1:1,000), Myc (Cell Signaling Technology, #2278, 1:1,000), Flag (Cell Signaling Technology, #2368, 1:1,000).

Techniques: Infection, Activity Assay