Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Immunohistochemical staining of PELP1 in TNBC. Positive immunostaining of PELP1 mainly distributed in nuclei of tumor cells, no cytoplasmic staining was found ( a , b ). Low grade lymph node stage TNBC showed weak PELP1 nuclear expression ( a ), High grade lymph node stage TNBC showed strong PELP1 nuclear expression ( b ). PELP1 nuclear staining was absent in negative control ( c ). Bar = 50 μm.

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Immunohistochemical staining, Staining, Immunostaining, Expressing, Negative Control

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Correlation between PELP1 protein expression and clinicopathological variables in patients with TNBC

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Clinicopathological variables and outcomes of patients with TNBC. Kaplan–Meier survival curve showed that TNBC patients with positive lymph node metastasis had significantly reduced DFS ( a1 ) and OS ( a2 ); TNBC patients in stage III and IV also demonstrated significantly reduced DFS ( b1 ) and OS ( b2 ); PELP1 was not associated with DFS or OS in TNBC patients when observed independently, although patients in the high PELP1 group demonstrated a trend of reduced DFS ( c1 ) and OS ( c2 ), compared with those in the low PELP1 group.

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Univariate analysis of DFS and OS according to clinicopathological variables

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: PELP1 protein expression and patients’ outcome in subgroups of TNBC. Kaplan–Meier survival curve showed that, in the tumor size ≤ 2 cm subgroup, patients with high PELP1 expression had significantly shorter DFS ( a1 ); in the high Ki-67 LI subgroups, patients with high PELP1 expression have significantly shorter DFS ( b1 ) and OS ( b2 ).

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Univariate analysis of DFS and OS according to PELP1 protein expression in different subgroups

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Combining PELP1 status and Ki-67 LI as a prognostic biological marker. Kaplan–Meier survival curve showed that, combination of PELP1 status with Ki-67 status was significantly correlated with DFS ( a1 ) and OS ( a2 ) in patients with TNBC; patients with TNBC in PELP1/Ki-67 double high group had significantly reduced DFS ( b1 ) and OS ( b2 ) compared with others.

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques: Marker

Journal: BMC Cancer

Article Title: Prognostic significance of proline, glutamic acid, leucine rich protein 1 (PELP1) in triple-negative breast cancer: a retrospective study on 129 cases

doi: 10.1186/s12885-015-1694-y

Figure Lengend Snippet: Multivariate analysis of DFS and OS according to clinical pathological variables

Article Snippet: Polyclonal antibody against PELP1 (Cat. IHC-00013, Bethyl Laboratories, Inc. Montgomery, AL, USA) was applied at an optimized dilution of 1:200 at 4 °C overnight.

Techniques:

Journal: Nature communications

Article Title: Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

doi: 10.1038/s41467-022-34610-0

Figure Lengend Snippet: Fig. 3 | Structural basis of PELP1-PELP1 dimerization. a Schematic representation of PELP1’s Rix1 domain with localization of LxxLL and PxxP motifs. Specific motifs involved in PELP1 dimerization, or residing close to dimer interfaces, are noted below the schematic. b Bottom view of segmented cryo-EM density showing two symmetric PELP1 dimerization interfaces between LM81-LM12-LM22 (superscript denotes specific protomer). c Model zoom of LM81-LM12-LM22 dimer interface exhibiting a hydrophobic environment produced by mainly leucine residues from LxxLL motifs. d Top view of cryo-EM density showing a single symmetric PELP1 dimerization interface between LM11 and α-helix 22 of each PELP1 protomer. e Model zoom of LM111+2 - α221+2 interface showing contributing leucine residues to another hydrophobic interface environment.

Article Snippet: Membranes were incubated overnight at 4 °C with ERα antibody (1/500, Millipore #06-935), PELP1 antibody (1/5000, Bethyl Labs #A300-180A-M) or WDR18 antibody (1/125, Sigma #HPA050200) and imaged using Azur Biosystem c600 imager.

Techniques: Cryo-EM Sample Prep, Produced

Journal: Nature communications

Article Title: Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

doi: 10.1038/s41467-022-34610-0

Figure Lengend Snippet: Fig. 5 | PELP1’s solvent-exposed LxxLL motifs are incompatible with steroid receptor binding. a Schematic representation of PELP1’s Rix1 domain with locali- zation of LxxLL and PxxP motifs. Motifs illustrated in model views are noted below the schematic (solvent motif labels colored blue). b Model view of a single PELP1 Rix1 domain protomer with solvent-exposed LxxLL motifs colored magenta. c, d, e Individual zooms of each solvent LxxLL motif with residue positions dis- played. Solvent face is illustrated in each panel with dashed line for spatial orien- tation of the motifs. The invariant leucine residues required for AF-2 binding of SRs are buried away from the solvent face, rendering them inaccessible for SR binding.

Article Snippet: Membranes were incubated overnight at 4 °C with ERα antibody (1/500, Millipore #06-935), PELP1 antibody (1/5000, Bethyl Labs #A300-180A-M) or WDR18 antibody (1/125, Sigma #HPA050200) and imaged using Azur Biosystem c600 imager.

Techniques: Solvent, Binding Assay, Residue

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 3: Depletion of LAS1L-interacting proteins induces a p53-dependent G1 cell cycle arrest. (A) Cell cycle profiles of HCT116 cells transfected with control, LAS1L, PELP1, TEX10, NOL9, SENP3, and WDR18 siRNA. Seventy-two hours after transfection the cells were pulse-labeled with BrdU for 30 min, stained with propidium iodide, and analyzed by flow cytometry to determine the percentage of cells in G1 and S phase. Error bars indicate SD from triplicate experiments. (B) Western blot analysis of the siRNA-transfected cells from (A) with specific antibodies against p53, p21, and β-actin. (C) Total RNA was extracted from the siRNA-transfected cells from panel (A), and knockdowns were confirmed by qRT-PCR with gene-specific primers. Relative mRNA levels for each gene-specific primer were normalized to β-actin. Error bars indicate SD from triplicate experiments.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Transfection, Control, Labeling, Staining, Flow Cytometry, Western Blot, Quantitative RT-PCR

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 2: LAS1L associates with PELP1, TEX10, WDR18, RanBP5, NOL9, and SENP3. (A) LAS1L was immunoprecipitated (IP) with an anti-LAS1L–specific antibody from HEK 293T cell lysates. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate; *, presence of an unspecific band. (B) PELP1 was immunoprecipitated (IP) with an anti-PELP1 antibody from HEK 293T cell lysates. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate. (C) HEK 293T cells were transfected with nontargeting control (represented by the letter “C”) or LAS1L siRNA for 48 h. Cells were lysed and immunoprecipitated with an anti-PELP1 antibody. Proteins associating with PELP1 in the absence of LAS1L were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate; *, presence of an unspecific band; #, an IgG band.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Negative Control, Transfection, Control

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 4: LAS1L-associated proteins localize to the nucleolus. Immunofluorescence analysis of U2OS cells with complex protein- specific antibodies. Cells were preextracted with 0.1% Triton, fixed, and immunostained with anti-LAS1L, PELP1, TEX10, NOL9, and WDR18 antibodies (green). Colocalization with SENP3 was confirmed using an anti-SENP3 antibody (red). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all three panels.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Immunofluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 6: LAS1L and NOL9 interact with the mammalian Rix1 complex on pre-60S ribosomal particles. Nuclear extracts from HCT116 cells were fractionated by centrifugation on a 10–30% sucrose gradient. Fractions were collected, and the optical density was measured at 260 nm (A260). Based on the A260 profile, fractions corresponding to free nuclear proteins (1, 2, and 3), pre-40S ribosomal particles (8, 9, and 10), and pre-60S ribosomal particles (12, 13, and 14) were then combined and immunoprecipitated (IP) with rabbit IgG (A) as a negative control or with PELP1 (B) or LAS1L (C) antibodies. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). WCL, whole-cell lysate; *, presence of an unspecific band; #, an IgG band.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Centrifugation, Immunoprecipitation, Negative Control, SDS Page, Western Blot

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 9: SENP3 is necessary for LAS1L and PELP1 nucleolar localization. (A) HCT116 cells were transfected with control or SENP3 siRNA for 72 h. Cells were fixed and immunostained with anti-LAS1L (green) and anti-SENP3 (red) antibodies. DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (B) HCT116 cells were transfected with control or SENP3 siRNA for 72 h. Cells were fixed and immunostained with anti-PELP1 (green) and anti-SENP3 (red) antibodies. DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (C) Cells were transfected with a Control (−) or SENP3 (+) siRNA for 72 h. Lysates were then immunoprecipitated with rabbit IgG (as negative control) or PELP1 antibody. Coprecipitating proteins were separated on SDS–PAGE and analyzed by Western blotting using specific antibodies (indicated on the left). WCL, whole-cell lysate; *, presence of an unspecific band; #, an IgG band.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Transfection, Control, Staining, Immunoprecipitation, Negative Control, SDS Page, Western Blot

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 8: PELP1 requires active Pol I transcription for nucleolar localization. (A) U2OS cells were treated with either DMSO or 20 nM actinomycin D for 2 h. Cells were fixed and immunostained with an anti-PELP1 antibody (green) and an anti-UBF1 antibody (red). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of both panels. (B) Cells were treated with DMSO (−) or actinomycin D (+) as in (A), and lysates were immunoprecipitated (IP) with an anti-PELP1 antibody. Associated proteins were separated on SDS–PAGE and analyzed by Western blotting with specific antibodies (indicated on the left). Normal rabbit IgG was used as negative control. WCL, whole-cell lysate.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Staining, Immunoprecipitation, SDS Page, Western Blot, Negative Control

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 10: NPM1 is required for LAS1L and PELP1 nucleolar localization. (A) HCT116 cells were transfected with control or NPM1 siRNA for 48 h. Subcellular localization of LAS1L was determined by immunofluorescence analysis using a LAS1L antibody (green). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (B) HCT116 cells were transfected with control or NPM1 siRNA for 48 h. Subcellular localization of PELP1 was determined by immunofluorescence analysis using a PELP1 antibody (green). DNA was visualized by staining with Hoechst 33342 (blue). Scale is representative of all panels. (C) Knockdowns of NPM1 for (A) and (B) were confirmed by Western blotting using specific antibodies (indicated on the left). An anti-CDK2 antibody was used as loading control. (D) Cells were transfected with a control (“C”) or NPM1 siRNA for 72 h. Lysates were then immunoprecipitated with rabbit IgG (as negative control) or PELP1 antibody. Coprecipitating proteins were separated on SDS–PAGE and analyzed by Western blotting using specific antibodies (indicated on the left). WCL, whole-cell lysate.

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Transfection, Control, Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Negative Control, SDS Page

Journal: Molecular Biology of the Cell

Article Title: LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis

doi: 10.1091/mbc.e11-06-0530

Figure Lengend Snippet: FIGURE 11: LAS1L and PELP1 are modified by SUMO in an SENP3-dependent manner. (A) HEK 293T cells were transfected with FLAG-LAS1L (+) plus either empty vector (−) or plasmids expressing 6xHis-tagged SUMO-1 or SUMO-3 (+). Forty-eight hours after transfection, SUMOylated proteins were pulled down from cell lysates using Ni-NTA agarose beads. Eluates were analyzed on SDS–PAGE and by Western blotting with the indicated antibodies. (B) HEK 293T cells were transfected with either control (−) or SENP3 (+) siRNA. The next day, cells were transfected with empty vector (−) or a 6xHis-tagged SUMO-3 (+) expressing plasmid. Forty- eight hours after transfection, pulldowns and protein analyses were performed as in (A). (C) HEK 293T cells were transfected with either control (−) or NPM1 (+) siRNA. The next day, cells were transfected with empty vector (−) or a plasmid expressing 6xHis-tagged SUMO-3 (+). Forty- eight hours after transfection, pulldowns and protein analyses were performed as in (A).

Article Snippet: The following antibodies were used: LAS1L (Sigma-Aldrich), PELP1 (Bethyl Laboratories, Montgomery, TX), NOL9 (ProteinTech), RanPB5 (Sigma-Aldrich), NPM1 (Santa Cruz Biotechnology, Santa Cruz, CA), TEX10 (ProteinTech), SENP3 (Santa Cruz Biotechnology), NOL9 (Abgent, San Diego, CA), p53 (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), CDK2 (Santa Cruz Biotechnology), RPL11 (Sigma-Aldrich), RPL26 (Sigma-Aldrich), and p21 (PharMingen, BD Biosciences, San Diego, CA).

Techniques: Modification, Transfection, Plasmid Preparation, Expressing, SDS Page, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Generation and characterization of PELP1 FBKO mice. (A) Schematic representation of endogenous (WT) and recombined locus mPELP1. (B) Confirmation of targeted clones by Southern blotting of genomic DNA. (C) Tail DNA PCR confirmation of bitransgenic PELP1 FBKO mice expressing CaMKIIα-Cre and loxpPELP1. (D) Confirmation of excision of floxed region in hippocampus and cortex of FLOX control and PELP1 FBKO mice using genomic PCR. (E) Immunohistochemical analysis of coronal sections of hippocampus, cortex, and hindbrain of control and PELP1 FBKO mice. (F) Lysates of hippocampus, cortex and cerebellum collected from control (PELP1 loxp/loxp), heterozygous (PELP1 loxp/−, CaMKIIαCre+/−), and homozygous PELP1 FBKO (PELP1 loxp/loxp, CaMKIIα-Cre+/−) mice were subjected to Western blotting with PELP1 antibody. β-actin was used as a loading control. (G) The data were quantified using Image J software and normalized to β-actin. C, Cre; F, FLOX. Bar graphs represent mean ± SEM. **P < 0.01.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Clone Assay, Southern Blot, Expressing, Immunohistochemical staining, Western Blot, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: E2 activation of extranuclear survival signaling and inhibition of proapoptotic JNK signaling is significantly attenuated in PELP1 FBKO mice. (A–D) Hippocampal lysates obtained from sham, sham E2, and at 10 min, 30 min, and 3 h after GCI/reperfusion from placebo and E2-treated ovariectomized female FLOX control mice (A and C) and PELP1 FBKO mice (B and D) were subjected to Western blotting. Blots were probed with phosphor- and total ERK1/2, Akt, and JNK antibodies. GAPDH was used as internal control. Pla = placebo; R = reperfusion. Bar graphs represent mean ± SEM. *P < 0.05; **P < 0.01 versus sham. n = 4–5 mice per group.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Activation Assay, Inhibition, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Immunohistochemistry results confirm that PELP1 FBKO mice exhibit reduced activation of extranuclear survival signaling with E2. Immunofluorescence staining of NeuN, p-Src, p-ERK1/2, and p-Akt in hippocampus CA1 at 30 min after GCI/reperfusion in FLOX control (A) and PELP1 FBKO mice (B). Pla = placebo. Results are representative of staining observed in five (n = 5) individual animals per group.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Immunohistochemistry, Activation Assay, Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Antisense oligonucleotide knockdown of PELP1 reverses E2-mediated effects on phosphorylation of Akt and JNK at 3h after GCI in the rat. MS or PELP1 AS oligonucleotides (10 μL, 10 nmol) were administered into the lateral cerebroventricle of adult ovariectomized rats every 24 h beginning 3 d before GCI and 1 h before GCI. E2 was administered by minipumps implanted subcutaneously at time of ovariectomy and left in place until the end of the experiment. The minipumps produce low diestrus levels (15–20 pg/mL) of E2 in the bloodstream. Western blot analysis results are shown. Note that black dotted lines between bands indicate representative bands that were noncontiguous. The results demonstrate a robust decrease of PELP1 levels in the hippocampal CA1 region of AS-treated rats versus MS-treated rats. Note also that PELP1 AS treatment reverses the E2-mediated effects upon Akt and JNK activation/phosphorylation after GCI (compare E2+AS to E2+MS). P < 0.05 between MS and AS treated groups. n = 5 per group.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Western Blot, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Duo-Link II in situ proximity ligation assay reveals that PELP1 interacts with ERα, PI3K regulatory subunit p85, and Src in vivo in the hippocampal CA1 region at 30 min after GCI, and that estradiol (E2) increases the interaction of PELP1 with these factors. Orange dots indicate protein interaction between PELP1 and ERα, P85, or Src. Tissue sections derived from sham, placebo, and E2 treated mice were blocked in 5% (vol/vol) goat serum for 1 h and incubated overnight with appropriate primary antibodies at 4 °C. Slides were then incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes for 1 h at 37 °C. Ligation and amplification was carried out using the Duolink detection reagent kit according to manufacturer’s protocol. Images were captured using confocal microscope. (Scale bar, 10 μm.)

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: In Situ, Proximity Ligation Assay, In Vivo, Derivative Assay, Incubation, Ligation, Amplification, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: E2-mediated enhancement of p-GSK3β and reduction of p-β-catenin is significantly attenuated in PELP1 FBKO mice. (A–D) Hippocampal lysates obtained from sham, sham E2, and at 10 min, 30 min, and 3 h after GCI/reperfusion from placebo and E2 treated ovariectomized female FLOX control mice (A and C) and PELP1 FBKO mice (B and D) were subjected to Western blotting to detect changes in the levels of p-GSK3β and p-β-catenin. GAPDH was used as an internal control. Representative blots show the attenuation of E2-mediated effects in PELP1 FBKO mice. Pla = placebo; R = reperfusion (C and D). Bar graphs represent mean ± SEM. *P < 0.05; **P < 0.01 versus sham. n = 4–5 mice per group.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Immunohistochemistry results confirm that PELP1 FBKO mice show a reduction in E2-mediated inactivation of p-GSK3β and activation of β-catenin. Immunofluorescence staining of p-GSK3β and p-β-catenin in hippocampus CA1 at 30 min after GCI/reperfusion in FLOX (A) and PELP1 FBKO (B) mice. Pla = placebo; R = reperfusion. Results are representative of staining observed in five individual animals per group.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Immunohistochemistry, Activation Assay, Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: PELP1 is a novel substrate of GSK3β. Hippocampal lysates collected 24 h after GCI reperfusion were subjected to immunoprecipitation with PELP1 or IgG antibody. Immunoprecipitates were pulled down by adding dynabeads protein A. Immunoprecipitates were subjected to SDS/PAGE, followed by mass spectrometry analysis. (A) List of unique PELP1-interacting proteins. (B) Forebrain lysates were subjected to immunoprecipitation or GST pull-down assay, and PELP1 interaction with GSK3β was confirmed by Western blotting. (C) Forebrain lysates were used in pull-down assays using GST or various deletion fragments of PELP1, and interaction of PELP1 deletions with GSK3β was analyzed by Western blotting. (D) GST-tagged PELP1 deletions and full-length GST-PELP1 were incubated with purified GSK3β enzyme in kinase buffer with [γ32-P] ATP and 100 μM cold ATP for 30 min at 30 °C, and the phosphorylation of PELP1 fragments was analyzed by PhosphorImager.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Pull Down Assay, Western Blot, Incubation, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: SDS/PAGE analysis of expression of various GST-PELP1 deletion fragments used in the study. Bands were visualized by Ponceau S staining. Asterisk points to the GST-PELP1 deletion protein band.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: SDS Page, Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Characterization of p-Thr745 PELP1 antibody. (A) Location of GSK3β binding and phosphorylation sites in PELP1. (B) Conservation of peptide sequence that contains the GSK3β phosphorylation site used for antibody preparation. (C) Western blots of hippocampus lysates (from two different mice) were probed with the Thr745 phospho-PELP1 antibody in the presence of control or phosphopeptide.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Binding Assay, Sequencing, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: GSK3β phosphorylation of PELP1 results in reduced stability of PELP1. (A) Tissue sections derived from sham, placebo, and E2-treated mice at 24 h after GCI were blocked in 5% (vol/vol) goat serum for 1 h and incubated overnight with appropriate primary antibodies at 4 °C. Slides were then incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes for 1 h at 37 °C. Ligation and amplification were carried out using the Duolink detection reagent kit according to manufacturer’s protocol. Images were captured using a confocal microscope. (B) Glioblastoma (U87) and neuroblastoma (SH-SY-5Y) cells were treated with GSK3β inhibitor lithium chloride, and lysates were collected at the indicated periods. Lysates were subjected to Western blot analysis with p745PELP1 and total PELP1 antibodies. (C) Coronal brain sections collected from sham, placebo, and E2-treated FLOX mice 6 d after GCI reperfusion were subjected to immunofluorescence for PELP1 antibody.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Derivative Assay, Incubation, Ligation, Amplification, Microscopy, Western Blot, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: PELP1 FBKO mice have alterations in E2-induced transcriptome. Total RNA was isolated from ovariectomized FLOX control mice and PELP1 FBKO mice (implanted with E2 mini pumps) that were subjected to GCI and killed at 24 h reperfusion. Hippocampi samples were subjected to RNA-seq analysis, as detailed in Methods. Differential expression analysis was performed by DEseq, and significant genes with at least twofold change were chosen for analysis. (A) Representative heat map of differentially expressed genes from two groups. (B) Differentially expressed genes with greater than twofold change in expression were further analyzed by ingenuity pathway analysis. The top canonical pathways are shown. (C) Selected differentially expressed genes were further validated by quantitative RT-PCR; RNA-seq expression values are plotted side by side for comparison.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Isolation, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: PELP1 FBKO mice have alterations in activation of various networks. Differentially expressed genes identified in RNA-seq analysis with greater than twofold change in expression were further analyzed by ingenuity pathway analysis. The top five associated network functions (A) and top five biological functions (B) are shown.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Activation Assay, RNA Sequencing Assay, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Gene interaction networks of differentially expressed genes between FLOX-E2 and PELP1-FBKO-E2 groups. Ingenuity pathway analysis of interaction of differentially expressed genes identified several networks. (A–E) Top five networks are shown. (A) Respiratory system development and function, connective tissue development and function, embryonic development. (B) Cellular assembly and organization, neurological disease, inflammatory disease. (C) Lipid metabolism, molecular transport, small molecule biochemistry. (D) Dermatological diseases and conditions, inflammatory disease, neurological disease. (E) Cell signaling, molecular transport, nucleic acid metabolism. A line represents the biological relationship between molecules, with solid lines representing direct interactions and the dashed lines representing indirect interactions. Genes that were up-regulated or down-regulated in PELP1-FBKO-E2 group were shown in red and green, respectively.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: PELP1 FBKO mice display diminished E2-induced neuroprotection: Coronal brain sections collected 6 d after GCI-reperfusion from sham, placebo, and E2-treated female ovariectomized FLOX control and PELP1 FBKO mice were subjected to cresyl violet and NeuN immunostaining, as described in Methods. (A) Representative Image of hippocampal CA1 region showing significant neuroprotection with E2 treatment, as evident from the significant increase in the number of surviving neurons compared with placebo in FLOX control mice. (B) Surviving neuronal counts of NeuN-positive neurons are shown. *P < 0.05 versus sham; #P < 0.05 versus placebo; $P < 0.05 versus FLOX placebo. (C) Spatial learning and memory were assessed in sham, placebo, and E2-treated female ovariectomized FLOX and PELP1 FBKO mice that were subjected to GCI, using the Barnes maze test, as described in Methods. (D) Time spent in each quadrant was quantified for all treatment groups. Data analysis indicated that PELP1 FBKO mice exhibited impaired E2-mediated spatial learning and memory. Data represent mean ± SE, n = 4–7 mice per group. *P < 0.05 and **P < 0.01.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Antisense oligonucleotide knockdown of PELP1 in the ovariectomized rat reverses E2-mediated neuroprotection after global cerebral ischemia. (A) Representative photomicrographs depict hippocampal CA1 neurons immunopositive for the neuronal nuclear marker NeuN 7 d after global cerebral ischemia. The experimental design is described in Fig. S2. PELP1 knockdown efficiency is also shown in Fig. S2. Note that E2-treated PELP1 AS knockdown rats had significantly fewer surviving NeuN-positive neurons compared with E2-treated MS animals after GCI, indicating a loss of E2 neuroprotection after PELP1 knockdown. Quantitative summary of data (means ± SE; n = 5–7 per group) is expressed as a percentage of NeuN-positive neurons per 250-μm medial CA1 of E2+MS and E2+AS animals. ^ = P < 0.05 compared with E2+MS. Magnification 40×. (Scale bar, 50 μm.) (B) Representative cresyl violet staining in the hippocampal CA1 region 7 d after global cerebral ischemia. Quantitative summary of data (means ± SE; n = 5–7 per group) is expressed as a percentage of cresyl violet-positive neurons per 250-μm medial CA1 of E2+MS and E2+AS animals. ^ = P < 0.05 compared with E2+MS. Magnification 40×. (Scale bar, 50 μm.)

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Marker, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Hyperactivity and anxiety-like behavior was assessed in sham, placebo, and E2-treated female ovariectomized FLOX and PELP1 FBKO mice that were subjected to GCI, using the open field test. Two days after reperfusion, mice were placed in an open field for 5 min, and overall activity was monitored using a video tracker. The total distance traveled in the open field chamber was calculated. Data analysis indicates there was no significant difference between placebo and E2-treated groups of FLOX and PELP1 KO mice. ns = nonsignificant.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques: Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

doi: 10.1073/pnas.1516729112

Figure Lengend Snippet: Antisense oligonucleotide knockdown of PELP1 in the ovariectomized rat reverses E2 enhancement of cognitive function after GCI. (A) Latency trail and (B) probe trail in the Morris water maze. Representative tracks for latency trail (C) and probe trail (D), [a and f: sham; b and g: ischemia/reperfusion (I/R); c and h: E2; d and i: E2+MS; e and j: E2+PELP1-AS]. Note that E2-treated animals had significantly reduced latency times to find the hidden platform in the water maze (A and C) and spent more time in the quadrant where the platform was located compared with I/R animals (B and D). MS oligos had no effect on E2 enhancement of cognitive function. PELP1 AS oligo knockdown markedly attenuated E2’s effect to decrease latency to find the platform (A and C) and increase time spent in the quadrant where the platform was located (B and D). The experimental design is as described in Fig. S2. PELP1 AS knockdown efficiency is also shown in Fig. S2. *P < 0.05 versus I/R group, #P < 0.05 versus E2+MS or E2+AS, n = 5–6.

Article Snippet: The p745-PELP1-phospho antibody was custom generated and affinity purified by Open Biosystems (Thermo-Fisher Scientific), using phospho-PELP1 Thr-745 peptide sequence (LAPSGpTPPP).

Techniques:

Journal: Cell reports

Article Title: TRMT1L-catalyzed m 2 2 G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.

doi: 10.1016/j.celrep.2024.115167

Figure Lengend Snippet: Figure 1. TRMT1L interacts with components of the Rix1 60S biogenesis complex in the nucleolus (A) Mass spectrometry analysis of PELP1-associated complexes. (B and C) (B) Western blot analysis of endogenous PELP1 or (C) TRMT1L immunoprecipitated. Asterisk indicates the presence of the IgG cross-reactivity band. (D) Schematic representation of TRMT1L and TRMT1 proteins. (E) Immunofluorescence detection of endogenous TRMT1L. Scale bar, 20 mm (F) Inhibition of rRNA transcription by CX-5461 impairs TRMT1L nucleolar localization in U2OS cells. Scale bar, 20 mm. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-PELP1 Antibody Bethyl Cat#A300-180A; RRID: AB_242526 Rabbit anti-TRMT1L Antibody Bethyl Cat#A305-120A; RRID: AB_2631515 Mouse anti-TRMT1L Antibody Sigma-Aldrich Cat#SAB1408048; RRID: AB_10741695 Rabbit anti-SENP3 Antibody Cell Signaling Technology Cat#5591; RRID: AB_10694546 Rabbit anti-WDR18 Antibody Castle et al.25 N/A Mouse anti-NPM1 Antibody Santa Cruz Biotechnology Cat#sc-56622; RRID: AB_784888 Mouse anti-uL5 (RPL11) Antibody Sigma-Aldrich Cat#SAB1402896; RRID: AB_10738164 Mouse c-Myc Antibody (9E10) Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 Rabbit anti-TRMT1 Antibody Bethyl Cat#A304-205A; RRID: AB_2620402 Rabbit anti-Fibrillarin Antibody Bethyl Cat#A303-891A; RRID: AB_2620241 Rabbit anti-DDX21 Antibody Bethyl Cat#A300-627A; RRID: AB_513601 Rabbit anti-N2,N2-dimethylguanosine (m2,2G) Antibody Abcam Cat#ab211488; RRID: AB_3532174 Rabbit IgG Normal Millipore Cat#NI01; RRID: AB_490574 Mouse anti-GAPDH Antibody Santa Cruz Biotechnology Cat#sc-47724; RRID: AB_627678 Mouse anti-puromycin antibody DSHB Cat#PMY-2A4; RRID: AB_2619605 Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa FluorTM 488 Thermo Fisher Scientific Cat#A-11001; RRID: AB_2534069 Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa FluorTM 594 Thermo Fisher Scientific Cat#A-11012; RRID: AB_2534079 Chemicals, peptides, and recombinant proteins Amersham Hybond N+ nylon membrane GE Healthcare RPN303B AMPure RNAClean XP beads Beckman Coulter A63987 AMV Reverse Transcriptase Promega M510A AMV Reverse Transcriptase 5X Reaction Buffer Promega M515A Aprotinin Fisher BP250310 AEBSF Fisher BP635-500 CX-5461 Cellagen Technologies C2954-2s DMEM (no glucose) Gibco 11966025 Exonuclease I NEB M0293S FastAP Thermosensitive Alkaline Phosphatase Thermo Scientific EF0651 Fetal bovine serum Hyclone SV30014.03 High glucose DMEM HyClone SH30022.01 Immun-Star AP substrate Bio-rad 1705018 Lipofectamine RNAi Max Invitrogen 13778150 Murine RNase inhibitor NEB M0314 Penicillin-Streptomycin Hyclone SH40003.01 Protease inhibitor cocktail Thermo Scientific 78437 Protinase K Invitrogen 46–7185 Protein G Dynabeads Invitrogen 10003D Puromycin Sigma P8833 pCp-Biotin Jena Bioscience NU-1706-Bio (Continued on next page) 18 Cell Reports 44, 115167, January 28, 2025

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Inhibition

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation *

doi: 10.1074/mcp.M116.064741

Figure Lengend Snippet: HIN domain of IFIX mediates its interaction with the 5FMC complex. A, validating IFIX co-interaction with SENP3 by reciprocal IP. B, IFIX interacts with 5FMC components LAS1L and SENP3 through its HIN domain. Forward IPs using GFP antibody were performed in cells transfected with IFIX constructs full-length (FL), IFIX Pyrin domain (PY), and IFIX HIN200 domain (HIN) in pEGFP. Inputs (1.5%), elutions (20%), and isolated IFIX constructs (IP, 20%) were blotted for LAS1L, SENP3, and GFP. C, IF microscopy in IFIX-GFP and EGFP control 293 cells showing a redistribution of 5FMC protein LAS1L in infected cells (white arrows), m.o.i.: 5, 4 hpi. Co-localization of IFIX and LAS1L is pronounced in uninfected cells (for PELP1, see supplemental Fig. S3). D, Levels of PELP1 and LAS1L are not reduced during HSV-1 infection. PELP1 and LAS1L levels were monitored by western blotting at 6hpi. ICP27 is marker for infection. M, mock. Microscopy images were taken at ×60 oil objective. Bar, 5 μm.

Article Snippet: Reagents The antibodies used for immunoaffinity purifications, Western blotting, and immunofluorescence were as follows: an in-house-generated α-green fluorescent protein (GFP) for IPs ( 25 ); α-GFP (Roche Applied Science) for Western blottings; α-PYHIN1 (Sigma-Aldrich); α-IFI16 antibodies (ab50004 and ab55328, Abcam, Cambridge, MA) used at a 1:1 mixture as described previously ( 22 ); α-ICP0 (H1A027-100, Virusys Corp., Taneytown, MD); α-ICP4 (sc-69809, Santa Cruz Biotechnology, Dallas, TX); α-ICP27 (sc-69806, Santa Cruz Biotechnology); α-ICP8 (sc-53329, Santa Cruz Biotechnology); α-tubulin (T6199, Sigma-Aldrich); α-PML (sc-9862, Santa Cruz Biotechnology); α-proteasome 20S core subunit (PW8155-0100, Enzo Life Sciences, Farmingdale, NY); α-HUWE1 (NB100-652, Novus, Littleton, CO); α-SENP3 (sc-67076, Santa Cruz Biotechnology); and α-LAS1L (ab140656) and α-PELP1 (sc-393534, Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Isolation, Microscopy, Infection, Western Blot, Marker

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation *

doi: 10.1074/mcp.M116.064741

Figure Lengend Snippet: IFIX inhibits viral gene transcription. A, confirmation of IFIX is overexpressed in stable HFFs. B, relative mRNA levels of viral genes ICP27 and ICP8 determined by quantitative PCR. Cells were infected with WT HSV-1 at an m.o.i. of 10 and collected at 6 hpi. Error bars represent S.D. of three biological replicates. Significance was determined by t test. *, p ≤ 0.05; **, p ≤ 0.005. C, IFIX-GFP localizes to viral genomes when its degradation is inhibited. Cells were treated with MG132 to block the proteasome during the course of 3- or 6-h infections. ICP4 is marker for viral genomes. Size bars, 5 μm. D, doubling times of HFFs stably expressing EGFP or IFIX-GFP. E, siRNA-mediated knockdown of 5FMC components PELP1 and SENP3 impact HSV-1 progeny titers in human fibroblasts. EGFP or IFIX-GFP HFFs were transfected with the indicated siRNAs. Cells were infected with WT HSV-1 at an m.o.i. of 10 at 24 h post-transfection. N.T., non-targeted. Error bars indicate S.D. of two biological replicates in technical duplicate; significance was determined by t test. *, p ≤ 0.05; **, p ≤ 0.005. n.s., not significant. F and G, validation of knockdown efficiency of PELP1 and SENP3 by Western blotting.

Article Snippet: Reagents The antibodies used for immunoaffinity purifications, Western blotting, and immunofluorescence were as follows: an in-house-generated α-green fluorescent protein (GFP) for IPs ( 25 ); α-GFP (Roche Applied Science) for Western blottings; α-PYHIN1 (Sigma-Aldrich); α-IFI16 antibodies (ab50004 and ab55328, Abcam, Cambridge, MA) used at a 1:1 mixture as described previously ( 22 ); α-ICP0 (H1A027-100, Virusys Corp., Taneytown, MD); α-ICP4 (sc-69809, Santa Cruz Biotechnology, Dallas, TX); α-ICP27 (sc-69806, Santa Cruz Biotechnology); α-ICP8 (sc-53329, Santa Cruz Biotechnology); α-tubulin (T6199, Sigma-Aldrich); α-PML (sc-9862, Santa Cruz Biotechnology); α-proteasome 20S core subunit (PW8155-0100, Enzo Life Sciences, Farmingdale, NY); α-HUWE1 (NB100-652, Novus, Littleton, CO); α-SENP3 (sc-67076, Santa Cruz Biotechnology); and α-LAS1L (ab140656) and α-PELP1 (sc-393534, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction, Infection, Blocking Assay, Marker, Stable Transfection, Expressing, Transfection, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PELP1 Suppression Inhibits Colorectal Cancer through c-Src Downregulation

doi: 10.1155/2014/193523

Figure Lengend Snippet: PELP1 expression was upregulated inCRC. (a) Western blot revealed that PELP1 protein expression was higher in the CRC cell lines HT-29, HCC-2998, SW-620, HCT-15, and COLO205 than in the normal colorectal epithelium FHC. (b) Informatics data suggested that PELP1 mRNA expression was increased in these five CRC cell lines.

Article Snippet: After quantification by a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China), equal amounts of proteins (20 μ g to 25 μ g) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and processed for immunoblotting with a rabbit multiclonal antibody for PELP1 (diluted at 1 : 100, Santa Cruz) and c-Src (diluted at 1 : 500, Santa Cruz).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PELP1 Suppression Inhibits Colorectal Cancer through c-Src Downregulation

doi: 10.1155/2014/193523

Figure Lengend Snippet: PELP1 downregulation inhibited the CRC cell line HT-29 in vitro. (a) After transfection with shRNA, PELP1 protein expression was decreased by 90%. shRNA #3 was selected for further investigations. (b) After PELP1 silencing, the cell viability of HT-29 was inhibited. (c) PELP1 silencing inhibited the colony formation ability of HT-29 by 57.5% (lower panel). A representative colony formation assay is shown (upper panel). (d) PELP1 silencing inhibited the migration ability of HT-29 by 69.3% (lower panel). A representative migration assay is shown (upper panel). (e) PELP1 silencing inhibited the invasion ability of HT-29 by 58% (lower panel). A representative invasive assay is shown (upper panel). (f) Senescence was induced by PELP1 silencing in HT-29. A representative β -Gal assay is shown.

Article Snippet: After quantification by a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China), equal amounts of proteins (20 μ g to 25 μ g) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and processed for immunoblotting with a rabbit multiclonal antibody for PELP1 (diluted at 1 : 100, Santa Cruz) and c-Src (diluted at 1 : 500, Santa Cruz).

Techniques: In Vitro, Transfection, shRNA, Expressing, Colony Assay, Migration

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PELP1 Suppression Inhibits Colorectal Cancer through c-Src Downregulation

doi: 10.1155/2014/193523

Figure Lengend Snippet: PELP1 downregulation inhibited the CRC cell line HT-29 in nude mice xenograft assay. PELP1 silencing inhibited CRC growth in nude mice (a). A representative nude mice xenograft assay is shown in (b).

Article Snippet: After quantification by a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China), equal amounts of proteins (20 μ g to 25 μ g) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and processed for immunoblotting with a rabbit multiclonal antibody for PELP1 (diluted at 1 : 100, Santa Cruz) and c-Src (diluted at 1 : 500, Santa Cruz).

Techniques: Xenograft Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PELP1 Suppression Inhibits Colorectal Cancer through c-Src Downregulation

doi: 10.1155/2014/193523

Figure Lengend Snippet: PELP1 silencing suppressed CRC through c-Src downregulation. (a) PELP1 silencing was accompanied by the downregulation of c-Src mRNA as determined by quantitative RT-PCR. (b) PELP1 silencing was accompanied by c-Src protein downregulation as determined by western blot. (c) Decreased cell viability induced by PELP1 silencing was recovered by c-Src upregulation in HT-29. (d) Decreased colony formation ability was recovered by c-Src upregulation in HT-29. (e) Decreased migration ability was recovered by c-Src upregulation in HT-29. (f) Decreased invasion ability was recovered by c-Src upregulation in HT-29. (g) Induced senescence by PELP1 silencing was inhibited after c-Src upregulation.

Article Snippet: After quantification by a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China), equal amounts of proteins (20 μ g to 25 μ g) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and processed for immunoblotting with a rabbit multiclonal antibody for PELP1 (diluted at 1 : 100, Santa Cruz) and c-Src (diluted at 1 : 500, Santa Cruz).

Techniques: Quantitative RT-PCR, Western Blot, Migration