antibodies against srp9  (Proteintech)

Journal: RNA Biology

Article Title: The role of SRP9/SRP14 in regulating Alu RNA

doi: 10.1080/15476286.2024.2430817

Figure Lengend Snippet: Organization of signal recognition particle (SRP) components across different domains of life. RNA helices are numbered according to previously described nomenclature . Tertiary interactions as depicted previously between helices 3 and 4 are represented by a black curve and T 1 symbol . Tertiary interactions between helices 6 and 8 are represented by a black circle and T 2 symbol. The symbol U describes the U-turn overlapping the UGUNR motif and is indicative of the τ-junction in the mammalian 7SL RNA . SRP9/SRP14 heterodimer, Srp14p homodimer, and HBsu homodimer are positioned over the UGUNR motif . SRP68/SRP72 heterodimer is positioned over the 5e helix . SRP19 and Sec65p are positioned over the GNAR motif . SRP54, Srp54p, and ffh are positioned over the 8b helix . The positioning of Srp21p has not yet been clearly identified . (A) Mammalian complex depicted by the Homo sapiens SRP. 7SL RNA secondary structure is presented as previously shown and scaffolds the SRP9/SRP14 heterodimer, SRP68/SRP72 heterodimer, SRP19, and SRP54; (B) fungal complex represented by the Saccharomyces cerevisiae SRP. scR1 RNA secondary structure is displayed as previously shown and scaffolds the Srp14p homodimer, Srp21p, Srp68p/Srp72p heterodimer, Sec65p, and Srp54p; (C) Archaeal complex demonstrated by the Methanococcus jannaschii SRP. 7S RNA secondary structure is shown as previously presented and scaffolds SRP19, and SRP54; (D) gram-positive bacterial complex demonstrated by the Bacillus subtilis SRP. scRNA secondary structure is displayed as previously shown and scaffolds the HBsu homodimer, and Ffh; (E) gram-negative bacterial complex is represented by the Escherichia coli SRP. The ffs secondary structure is demonstrated as previously shown and scaffolds Ffh.

Article Snippet: Schematics of biomolecules (not to scale) depicting SRP9/SRP14 (red circles), DNA (double helix), Alu RNA (dimeric Alu RNA secondary structure), non-Alu RNA mRNA (cyan lines), and a ribosome (overlapping black oval and circle) are shown.

Techniques:

Journal: RNA Biology

Article Title: The role of SRP9/SRP14 in regulating Alu RNA

doi: 10.1080/15476286.2024.2430817

Figure Lengend Snippet: Life cycle of Alu RNA regulated by SRP9/SRP14. Schematics of biomolecules (not to scale) depicting SRP9/SRP14 (red circles), DNA (double helix), Alu RNA (dimeric Alu RNA secondary structure), non-Alu RNA mRNA (cyan lines), and a ribosome (overlapping black oval and circle) are shown. SRP9/SRP14 is distributed non-specifically across the DNA. i) SRP9/SRP14 interacts with nascent nuclear Alu RNA, in the context of non-coding RNA and mRNA; ii) non-coding Alu RNA is trafficked into the nucleolus by SRP9/SRP14; ii-b) Alu RNA embedded in mRNA is alternatively spliced depending on the absence or presence of SRP9/SRP14; iii) inside the nucleolus, Alu RNA undergoes post-transcriptional modifications including an N 6 -methyladenylation (m 6 A) and addition of a 3’ adenosine; iv) non-coding Alu RNA is processed into small cytoplasmic Alu RNA (scAlu RNA). The location of this process has not been determined, but is possible to occur in the nucleolus, as suggested by similar localization patterns of full-length and small cytoplasmic Alu RNA ; v) non-coding Alu RNA, scAlu RNA, and mRNA embedded Alu RNA are trafficked into the cytoplasm; vi) Alu RNA is brought in close proximity to ribosomes by SRP9/SRP14; vii) ribosome translating ORF2 protein performs retrotransposition on Alu RNA; viii) ribosomes involved in translation are directly regulated by Alu RNA; ix) Alu RNA induces stress granule assembly, including components such as stalled ribosomes; x) cytoplasmic Alu RNA is packaged into vesicles that are exocytosed into the extracellular matrix as exosomes; xi) cytoplasmic Alu RNA is packaged with retroviruses and excreted into the extracellular matrix.

Article Snippet: Schematics of biomolecules (not to scale) depicting SRP9/SRP14 (red circles), DNA (double helix), Alu RNA (dimeric Alu RNA secondary structure), non-Alu RNA mRNA (cyan lines), and a ribosome (overlapping black oval and circle) are shown.

Techniques:

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Comparison of patient prognoses (RFS and OS) in two SRP9 expression groups. In immunohistochemistry staining for SRP9, the tumors were divided into two groups: (A) Staining of ≤50% of the total number of nuclei in the tumor and (B) staining of >50% of the tumor area (magnification, ×40; scale bar, 50 µ m). The (C) RFS and (D) OS of the two SRP9 expression groups were compared. RFS, recurrence-free survival; OS, overall survival; SRP9, signal recognition particle 9.

Article Snippet: Sections were a thickness of 20 µ m. Immunohistochemistry using antibodies against SRP9 (cat. no. 11195-1-AP; RRID: AB_2239820; Proteintech Group, Inc.) ( ) and Ki-67 (cat. no. 27309-1-AP; Proteintech Group, Inc.) ( ) were performed using the excised specimens at concentrations of 1:200, and 1:2,500, respectively, and both were incubated at 4°C for 24 h. Normal goat serum blocking solution (cat. no. S-1000-20; Vector Laboratories, Inc.; Maravai Life Sciences) was used at a concentration of 1:200 for 60 min at room temperature for blocking, and anti-IgG (H+L), rabbit, goat-polyclonal secondary antibody (cat. no. BA-1000-1.5; Vector Laboratories Inc.; Maravai Life Sciences) was used at a concentration of 1:500 for 60 min at room temperature.

Techniques: Comparison, Expressing, Immunohistochemistry, Staining

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Immunocytochemistry to evaluate differences in nuclear translocation rates between pancreatic cancer lines cultured in normal and amino acid-deficient medium. (A) The pathway of methionine and tryptophan/niacin upstream of the cycle, which is deficient in the medium used in the present study. (B) The 'Cytoplasm stained with SRP9' and the 'Overlap area,' which are the staining covers of DAPI and SRP9 used in the 'S' and 'R' equations. The MiaPaCa pancreatic cancer cell line was cultured in regular medium for 24 h and then divided into four different media: MTN(+), M(−), TN(−) and MTN(−). After 24 h of incubation, SRP9 and nuclear immunocytochemistry tests were performed. (C) A total of 16 locations were randomly selected in one sample. The area of SRP9 (S all , defined as 'cytoplasm stained with SRP9') and the area where SRP9 and DAPI overlapped (S nuclei , defined as 'overlapping area') were measured to determine S (nuclear transfer rate=S nuclei /S all ), and the nuclear translocation rate expressed by the equation S was calculated. The nuclear transfer rate was defined as R [nuclear transfer rate=R nuclei /R all (R=S x fluorescence intensity)] by taking the ratio of the area of SRP9 multiplied by the fluorescence intensity. Representative images with (D) SRP9 fluorescence and (E) DAPI overlap. MiaPaCa cells were cultured in MTN(+), M(−), TN(−) or MTN(−), and the nuclear transfer rates were compared using the equations expressed as (F) S and (G) R. Error bars indicate standard error. ** P<0.01, *** P<0.001, **** P<0.0001 by Dunn test. Images of immunocyte staining were collected at ×40 magnification, and the scale bars indicate 100 µ m. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; NNMT, nicotinamide N-methyltransferase; NAD + , nicotinamide adenine dinucleotide; SRP9, signal recognition particle 9; MTN(+), consisting of L-amino acids, niacin and tryptophan; M(−), methionine-free medium; TN(−), tryptophan- and niacin-free medium; MTN(−), methionine-, tryptophan- and niacin-free medium.

Article Snippet: Sections were a thickness of 20 µ m. Immunohistochemistry using antibodies against SRP9 (cat. no. 11195-1-AP; RRID: AB_2239820; Proteintech Group, Inc.) ( ) and Ki-67 (cat. no. 27309-1-AP; Proteintech Group, Inc.) ( ) were performed using the excised specimens at concentrations of 1:200, and 1:2,500, respectively, and both were incubated at 4°C for 24 h. Normal goat serum blocking solution (cat. no. S-1000-20; Vector Laboratories, Inc.; Maravai Life Sciences) was used at a concentration of 1:200 for 60 min at room temperature for blocking, and anti-IgG (H+L), rabbit, goat-polyclonal secondary antibody (cat. no. BA-1000-1.5; Vector Laboratories Inc.; Maravai Life Sciences) was used at a concentration of 1:500 for 60 min at room temperature.

Techniques: Immunocytochemistry, Translocation Assay, Cell Culture, Staining, Incubation, Fluorescence

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Evaluation of nuclear translocation of artificially constructed SRP9 isoforms. (A) Variants of SRP9, v1 and v2. (B) MiaPaCa cells cultured in regular medium for 24 h were transfected with plasmids incorporating the genes of v1, v2 and the common parts of v1 and v2 and cultured for 48 h, after which fluorescence was observed under a microscope. (C) SRP9 expression was not observed in MOCK cells prior to transfection with the plasmids. The results of fluorescence in (D) v1, (E) v2, and (F) the common parts of v1 and v2. Images of immunocyte staining were collected at ×40 magnification, and the scale bars indicate 100 µ m. v1, variant 1; v2, variant 2; SRP9, signal recognition particle 9.

Article Snippet: Sections were a thickness of 20 µ m. Immunohistochemistry using antibodies against SRP9 (cat. no. 11195-1-AP; RRID: AB_2239820; Proteintech Group, Inc.) ( ) and Ki-67 (cat. no. 27309-1-AP; Proteintech Group, Inc.) ( ) were performed using the excised specimens at concentrations of 1:200, and 1:2,500, respectively, and both were incubated at 4°C for 24 h. Normal goat serum blocking solution (cat. no. S-1000-20; Vector Laboratories, Inc.; Maravai Life Sciences) was used at a concentration of 1:200 for 60 min at room temperature for blocking, and anti-IgG (H+L), rabbit, goat-polyclonal secondary antibody (cat. no. BA-1000-1.5; Vector Laboratories Inc.; Maravai Life Sciences) was used at a concentration of 1:500 for 60 min at room temperature.

Techniques: Translocation Assay, Construct, Cell Culture, Transfection, Fluorescence, Microscopy, Expressing, Staining, Variant Assay

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Functional evaluation of v1 and v2 by RIP sequencing. (A) Overview of RIP sequencing. The MiaPaCa cell line before transfection of the SRP9 (B) v1 and (C) v2 plasmids (MOCK cells) showed no AcGFP signal in both cases when observed using a fluorescent all-in-one microscope. Then, protein expression of (D) v1 and (E) v2 via AcGFP signal was confirmed following transfection with pIRES2-AcGFP1-NSL-SRP9V1 or pIRES2-AcGFP1-NSL-SRP9V2 plasmids. Results of the (F) heatmap, (G) volcano plot and (H) cluster analysis using the k-means algorithm for RIP1, RIP2, INPUT1 and INPUT2. Top pathways enriched in GSEA for (I) RIP1 + RIP2 vs. INPUT1 + INPUT2, (J) RIP1 vs. INPUT1, (K) RIP2 vs. INPUT2 and (L) RIP1 vs. RIP2. (M) In the GSEA, the KRAS signaling DN pathway was the most upregulated in the RIP samples for RIP1 + RIP2 vs. INPUT1 + INPUT2, RIP1 vs. INPUT1, and RIP2 vs. INPUT2. v1, variant 1; v2, variant 2; SRP9, signal recognition particle 9; RIP, RNA immunoprecipitation (sample); FDR, false discovery rate; GSEA, Gene Set Enrichment Analysis; DN, downregulated; NES, Normalized Enrichment Score; NOM, nominal.

Article Snippet: Sections were a thickness of 20 µ m. Immunohistochemistry using antibodies against SRP9 (cat. no. 11195-1-AP; RRID: AB_2239820; Proteintech Group, Inc.) ( ) and Ki-67 (cat. no. 27309-1-AP; Proteintech Group, Inc.) ( ) were performed using the excised specimens at concentrations of 1:200, and 1:2,500, respectively, and both were incubated at 4°C for 24 h. Normal goat serum blocking solution (cat. no. S-1000-20; Vector Laboratories, Inc.; Maravai Life Sciences) was used at a concentration of 1:200 for 60 min at room temperature for blocking, and anti-IgG (H+L), rabbit, goat-polyclonal secondary antibody (cat. no. BA-1000-1.5; Vector Laboratories Inc.; Maravai Life Sciences) was used at a concentration of 1:500 for 60 min at room temperature.

Techniques: Functional Assay, Sequencing, Transfection, Microscopy, Expressing, Variant Assay, Immunoprecipitation

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Comparison of patient prognoses (RFS and OS) in two SRP9 expression groups. In immunohistochemistry staining for SRP9, the tumors were divided into two groups: (A) Staining of ≤50% of the total number of nuclei in the tumor and (B) staining of >50% of the tumor area (magnification, ×40; scale bar, 50 µ m). The (C) RFS and (D) OS of the two SRP9 expression groups were compared. RFS, recurrence-free survival; OS, overall survival; SRP9, signal recognition particle 9.

Article Snippet: According to the Human Protein Atlas database ( https://www.proteinatlas.org ), patients with pancreatic cancer with high SRP9 expression tend to have poorer overall survival (OS).

Techniques: Comparison, Expressing, Immunohistochemistry, Staining

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Immunocytochemistry to evaluate differences in nuclear translocation rates between pancreatic cancer lines cultured in normal and amino acid-deficient medium. (A) The pathway of methionine and tryptophan/niacin upstream of the cycle, which is deficient in the medium used in the present study. (B) The 'Cytoplasm stained with SRP9' and the 'Overlap area,' which are the staining covers of DAPI and SRP9 used in the 'S' and 'R' equations. The MiaPaCa pancreatic cancer cell line was cultured in regular medium for 24 h and then divided into four different media: MTN(+), M(−), TN(−) and MTN(−). After 24 h of incubation, SRP9 and nuclear immunocytochemistry tests were performed. (C) A total of 16 locations were randomly selected in one sample. The area of SRP9 (S all , defined as 'cytoplasm stained with SRP9') and the area where SRP9 and DAPI overlapped (S nuclei , defined as 'overlapping area') were measured to determine S (nuclear transfer rate=S nuclei /S all ), and the nuclear translocation rate expressed by the equation S was calculated. The nuclear transfer rate was defined as R [nuclear transfer rate=R nuclei /R all (R=S x fluorescence intensity)] by taking the ratio of the area of SRP9 multiplied by the fluorescence intensity. Representative images with (D) SRP9 fluorescence and (E) DAPI overlap. MiaPaCa cells were cultured in MTN(+), M(−), TN(−) or MTN(−), and the nuclear transfer rates were compared using the equations expressed as (F) S and (G) R. Error bars indicate standard error. ** P<0.01, *** P<0.001, **** P<0.0001 by Dunn test. Images of immunocyte staining were collected at ×40 magnification, and the scale bars indicate 100 µ m. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; NNMT, nicotinamide N-methyltransferase; NAD + , nicotinamide adenine dinucleotide; SRP9, signal recognition particle 9; MTN(+), consisting of L-amino acids, niacin and tryptophan; M(−), methionine-free medium; TN(−), tryptophan- and niacin-free medium; MTN(−), methionine-, tryptophan- and niacin-free medium.

Article Snippet: According to the Human Protein Atlas database ( https://www.proteinatlas.org ), patients with pancreatic cancer with high SRP9 expression tend to have poorer overall survival (OS).

Techniques: Immunocytochemistry, Translocation Assay, Cell Culture, Staining, Incubation, Fluorescence

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Evaluation of nuclear translocation of artificially constructed SRP9 isoforms. (A) Variants of SRP9, v1 and v2. (B) MiaPaCa cells cultured in regular medium for 24 h were transfected with plasmids incorporating the genes of v1, v2 and the common parts of v1 and v2 and cultured for 48 h, after which fluorescence was observed under a microscope. (C) SRP9 expression was not observed in MOCK cells prior to transfection with the plasmids. The results of fluorescence in (D) v1, (E) v2, and (F) the common parts of v1 and v2. Images of immunocyte staining were collected at ×40 magnification, and the scale bars indicate 100 µ m. v1, variant 1; v2, variant 2; SRP9, signal recognition particle 9.

Article Snippet: According to the Human Protein Atlas database ( https://www.proteinatlas.org ), patients with pancreatic cancer with high SRP9 expression tend to have poorer overall survival (OS).

Techniques: Translocation Assay, Construct, Cell Culture, Transfection, Fluorescence, Microscopy, Expressing, Staining, Variant Assay

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Functional evaluation of v1 and v2 by RIP sequencing. (A) Overview of RIP sequencing. The MiaPaCa cell line before transfection of the SRP9 (B) v1 and (C) v2 plasmids (MOCK cells) showed no AcGFP signal in both cases when observed using a fluorescent all-in-one microscope. Then, protein expression of (D) v1 and (E) v2 via AcGFP signal was confirmed following transfection with pIRES2-AcGFP1-NSL-SRP9V1 or pIRES2-AcGFP1-NSL-SRP9V2 plasmids. Results of the (F) heatmap, (G) volcano plot and (H) cluster analysis using the k-means algorithm for RIP1, RIP2, INPUT1 and INPUT2. Top pathways enriched in GSEA for (I) RIP1 + RIP2 vs. INPUT1 + INPUT2, (J) RIP1 vs. INPUT1, (K) RIP2 vs. INPUT2 and (L) RIP1 vs. RIP2. (M) In the GSEA, the KRAS signaling DN pathway was the most upregulated in the RIP samples for RIP1 + RIP2 vs. INPUT1 + INPUT2, RIP1 vs. INPUT1, and RIP2 vs. INPUT2. v1, variant 1; v2, variant 2; SRP9, signal recognition particle 9; RIP, RNA immunoprecipitation (sample); FDR, false discovery rate; GSEA, Gene Set Enrichment Analysis; DN, downregulated; NES, Normalized Enrichment Score; NOM, nominal.

Article Snippet: According to the Human Protein Atlas database ( https://www.proteinatlas.org ), patients with pancreatic cancer with high SRP9 expression tend to have poorer overall survival (OS).

Techniques: Functional Assay, Sequencing, Transfection, Microscopy, Expressing, Variant Assay, Immunoprecipitation

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Comparison of patient prognoses (RFS and OS) in two SRP9 expression groups. In immunohistochemistry staining for SRP9, the tumors were divided into two groups: (A) Staining of ≤50% of the total number of nuclei in the tumor and (B) staining of >50% of the tumor area (magnification, ×40; scale bar, 50 µ m). The (C) RFS and (D) OS of the two SRP9 expression groups were compared. RFS, recurrence-free survival; OS, overall survival; SRP9, signal recognition particle 9.

Article Snippet: Of these, high SRP9 expression tends to be related to a worse OS in patients with pancreatic cancer, according to the Human Protein Atlas database ( https://www.proteinatlas.org/ENSG00000143742-SRP9/pathology/pancreatic+cancer ).

Techniques: Comparison, Expressing, Immunohistochemistry, Staining

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Immunocytochemistry to evaluate differences in nuclear translocation rates between pancreatic cancer lines cultured in normal and amino acid-deficient medium. (A) The pathway of methionine and tryptophan/niacin upstream of the cycle, which is deficient in the medium used in the present study. (B) The 'Cytoplasm stained with SRP9' and the 'Overlap area,' which are the staining covers of DAPI and SRP9 used in the 'S' and 'R' equations. The MiaPaCa pancreatic cancer cell line was cultured in regular medium for 24 h and then divided into four different media: MTN(+), M(−), TN(−) and MTN(−). After 24 h of incubation, SRP9 and nuclear immunocytochemistry tests were performed. (C) A total of 16 locations were randomly selected in one sample. The area of SRP9 (S all , defined as 'cytoplasm stained with SRP9') and the area where SRP9 and DAPI overlapped (S nuclei , defined as 'overlapping area') were measured to determine S (nuclear transfer rate=S nuclei /S all ), and the nuclear translocation rate expressed by the equation S was calculated. The nuclear transfer rate was defined as R [nuclear transfer rate=R nuclei /R all (R=S x fluorescence intensity)] by taking the ratio of the area of SRP9 multiplied by the fluorescence intensity. Representative images with (D) SRP9 fluorescence and (E) DAPI overlap. MiaPaCa cells were cultured in MTN(+), M(−), TN(−) or MTN(−), and the nuclear transfer rates were compared using the equations expressed as (F) S and (G) R. Error bars indicate standard error. ** P<0.01, *** P<0.001, **** P<0.0001 by Dunn test. Images of immunocyte staining were collected at ×40 magnification, and the scale bars indicate 100 µ m. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; NNMT, nicotinamide N-methyltransferase; NAD + , nicotinamide adenine dinucleotide; SRP9, signal recognition particle 9; MTN(+), consisting of L-amino acids, niacin and tryptophan; M(−), methionine-free medium; TN(−), tryptophan- and niacin-free medium; MTN(−), methionine-, tryptophan- and niacin-free medium.

Article Snippet: Of these, high SRP9 expression tends to be related to a worse OS in patients with pancreatic cancer, according to the Human Protein Atlas database ( https://www.proteinatlas.org/ENSG00000143742-SRP9/pathology/pancreatic+cancer ).

Techniques: Immunocytochemistry, Translocation Assay, Cell Culture, Staining, Incubation, Fluorescence

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Evaluation of nuclear translocation of artificially constructed SRP9 isoforms. (A) Variants of SRP9, v1 and v2. (B) MiaPaCa cells cultured in regular medium for 24 h were transfected with plasmids incorporating the genes of v1, v2 and the common parts of v1 and v2 and cultured for 48 h, after which fluorescence was observed under a microscope. (C) SRP9 expression was not observed in MOCK cells prior to transfection with the plasmids. The results of fluorescence in (D) v1, (E) v2, and (F) the common parts of v1 and v2. Images of immunocyte staining were collected at ×40 magnification, and the scale bars indicate 100 µ m. v1, variant 1; v2, variant 2; SRP9, signal recognition particle 9.

Article Snippet: Of these, high SRP9 expression tends to be related to a worse OS in patients with pancreatic cancer, according to the Human Protein Atlas database ( https://www.proteinatlas.org/ENSG00000143742-SRP9/pathology/pancreatic+cancer ).

Techniques: Translocation Assay, Construct, Cell Culture, Transfection, Fluorescence, Microscopy, Expressing, Staining, Variant Assay

Journal: International Journal of Oncology

Article Title: Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality

doi: 10.3892/ijo.2024.5662

Figure Lengend Snippet: Functional evaluation of v1 and v2 by RIP sequencing. (A) Overview of RIP sequencing. The MiaPaCa cell line before transfection of the SRP9 (B) v1 and (C) v2 plasmids (MOCK cells) showed no AcGFP signal in both cases when observed using a fluorescent all-in-one microscope. Then, protein expression of (D) v1 and (E) v2 via AcGFP signal was confirmed following transfection with pIRES2-AcGFP1-NSL-SRP9V1 or pIRES2-AcGFP1-NSL-SRP9V2 plasmids. Results of the (F) heatmap, (G) volcano plot and (H) cluster analysis using the k-means algorithm for RIP1, RIP2, INPUT1 and INPUT2. Top pathways enriched in GSEA for (I) RIP1 + RIP2 vs. INPUT1 + INPUT2, (J) RIP1 vs. INPUT1, (K) RIP2 vs. INPUT2 and (L) RIP1 vs. RIP2. (M) In the GSEA, the KRAS signaling DN pathway was the most upregulated in the RIP samples for RIP1 + RIP2 vs. INPUT1 + INPUT2, RIP1 vs. INPUT1, and RIP2 vs. INPUT2. v1, variant 1; v2, variant 2; SRP9, signal recognition particle 9; RIP, RNA immunoprecipitation (sample); FDR, false discovery rate; GSEA, Gene Set Enrichment Analysis; DN, downregulated; NES, Normalized Enrichment Score; NOM, nominal.

Article Snippet: Of these, high SRP9 expression tends to be related to a worse OS in patients with pancreatic cancer, according to the Human Protein Atlas database ( https://www.proteinatlas.org/ENSG00000143742-SRP9/pathology/pancreatic+cancer ).

Techniques: Functional Assay, Sequencing, Transfection, Microscopy, Expressing, Variant Assay, Immunoprecipitation

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: The RNA component of SRP, 7SL, is synthesized in the nucleoplasm by RNA polymerase III, where its 3′ end is bound by protein La. Other maturation steps occur in the nucleolus and/or putatively the Cajal bodies (CBs) as indicated. Five of the six SRP protein subunits (SRP9, SRP14, SRP19, SRP68, and SRP72) are assembled in the nucleolus (see text for details). After export to the cytoplasm, the sixth subunit (SRP54) is assembled, aided by the SMN complex, and mature SRP is produced. The Alu and S domains of 7SL are indicated.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Synthesized, Produced

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D, E, F, G, H) For U2OS Flp-In T-REx cells (left column), the expression of the genes encoding GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), and GFP-SRP14 (D) was induced by addition of Dox (1 μg/ml) to the medium and incubation for the indicated time. (E, F, G, H) Same concentration of Dox was used to induce the expression of the genes encoding GFP-SRP19 (E), GFP-SRP72 (F), GFP-SRP9 (G), and GFP-SRP14 (H) in HEK293 Flp-In T-REx cells (right column). Amounts of each GFP-SRP protein and the corresponding endogenous protein were analyzed by SDS–PAGE and WB with specific antibodies. GAPDH or tubulin β was used as a loading control. The expression of a given GFP-tagged SRP construct generally led to a reduced level of the corresponding endogenous protein. To us, this suggests that cells somehow regulate overall amounts of SRP proteins via a mechanism that remains to be determined. (I, J) Proteins SRP14 and SRP72 exist in different isoforms. U2OS Flp-In T-REx cells were transfected for 48 h with siRNAs specific to the mRNA encoding the SRP14 protein (si-SRP14) (I) or the SRP72 protein (si-SRP72) (J). Control siRNAs targeting the mRNA encoding luciferase (si-Luc) were used as negative controls. Total extracts were prepared and analyzed by SDS–PAGE and WB. Tubulin β was used as a loading control. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Expressing, Incubation, Concentration Assay, SDS Page, Construct, Transfection, Luciferase, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D) Total extracts were produced from HEK293 Flp-In T-REx cells having expressed for 3 h one of the following proteins: GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), or GFP-SRP14 (D). IPs were carried out, in the presence (+) and absence (−) of RNase A, with GFP-Trap beads and either one of these extracts or an extract of parental HEK293 Flp-In T-REx cells (Control). The immunoprecipitate (IP) and a fraction of the total cell extract (5%) (Total) were analyzed by SDS–PAGE and WB with antibodies against the indicated proteins. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Produced, SDS Page, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D) IP-SILAC analyses performed on HEK293 Flp-In T-REx cells expressing GFP-SRP19 (A), GFP-SRP9 (C), or GFP-SRP14 (D) for 3 h or on U2OS Flp-In T-REx cells expressing GFP-SRP72 for 3 h (B). The graph displays the log 2 of the SILAC ratio (y-axis, specific IP versus control IP performed with parental Flp-In T-REx cells) as a function of signal abundance (x-axis, log 10 (intensity)/MW). Each dot represents a protein. The labeled dots were arbitrarily selected to highlight proteins relevant to this study and families of proteins (see Key below the graphs) associated with GFP-SRP proteins. Analysis of the functions of the associated proteins was performed with the Gene Ontology Resource and UniProt. The full hit list with Significance B values is given in Table S1. The indicated percentage of nucleolar proteins and/or proteins involved in ribosome biogenesis, as well as the one of ribosomal proteins, represents the percentages in the number of these classes of proteins among all the associated proteins with the GFP-SRP protein analyzed and with a SILAC ratio above 1.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Expressing, Labeling

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D) Synthesis of GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), and GFP-SRP14 (D) was induced for 12 h in U2OS Flp-In T-REx cells. Total extracts were prepared, and IPs were carried out with GFP-Trap beads on extracts from parental U2OS Flp-In T-REx cells (Control) and U2OS Flp-In T-REx cells expressing one of the GFP-SRP proteins. The immunoprecipitate (IP) and a fraction (5%) of the total protein extract (Total) were analyzed by SDS–PAGE and WB with antibodies against the indicated proteins. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Expressing, SDS Page, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D, E, F) Expression of the gene encoding GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), or GFP-SRP14 (E) in HEK293 Flp-In T-REx cells or GFP-SRP9 (D) or GFP-SRP14 (F) in U2OS Flp-In T-Rex cells was induced with Dox for 12 h. The subcellular localization of the indicated protein was then analyzed by direct fluorescence of the GFP tag. Nuclei were stained in blue with DAPI. Images were acquired with an epifluorescence microscope. GFP-SRP proteins are in green. Scale bar: 15 μm. (G, H) Expression of the genes encoding GFP-SRP19 and GFP-SRP72 was induced with Dox for 12 h in HEK293 Flp-In T-REx cells. Double IF experiments were performed with anti-coilin (a marker of CBs) and anti-NCL (a marker of nucleolus) antibodies. Images were acquired with a confocal microscope. GFP-SRP19 and GFP-SRP72 are in cyan, and their localization was determined by direct GFP fluorescence analysis. Coilin is in magenta, and NCL is in yellow. White arrows indicate the co-localization of the GFP-SRP19 or GFP-SRP72 protein with coilin. Scale bar: 7 μm.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Expressing, Fluorescence, Staining, Microscopy, Marker

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A) Venn diagram showing the intersection between nucleolar proteins and/or proteins involved in ribosome biogenesis present in the GFP-SRP9, GFP-SRP14, GFP-SRP19, and GFP-SRP72 interactomes as determined by IP-SILAC analysis. The diagram includes all the proteins associated with a SILAC ratio up to 1. The ones associated with at least 3 GFP-SRP proteins are listed in red. (B) IPs were carried out on U2OS Flp-In T-REx cell total extracts, using anti-SRP19 (upper panels) and anti-SRP9 (lower panels) antibodies bound to magnetic beads with recombinant protein A (Dynabeads Protein A). Beads alone were used as a negative control (Control). The immunoprecipitate (IP) and a fraction of the total cell extract (5%) (Total) were analyzed by SDS–PAGE. The indicated proteins were revealed by WB. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: The following antibodies were used: anti-SRP9 (11195-1-AP; Proteintech) rabbit polyclonal, anti-SRP14 (11528-1-AP; Proteintech) rabbit polyclonal, anti-SRP19 (16033-1-AP; Proteintech) rabbit polyclonal, anti-SRP54 (610940; BD Bioscience) mouse monoclonal, anti-SRP68 (11585-1-AP; Proteintech) rabbit polyclonal, anti-SRP72 (AP17766PU-N; OriGene) rabbit polyclonal, anti-GFP (GTX113617; Genetex) rabbit polyclonal, anti-coilin (ab11822; Abcam) mouse monoclonal, anti-coilin (A303-759A; BETHYL) rabbit polyclonal, anti-NCL (ab136649; Abcam) mouse monoclonal, anti-FBL (72B9) mouse monoclonal , anti-NPM1 (ab40696; Abcam) mouse monoclonal, anti-BiP/GRP78 (ab21685; Abcam) rabbit polyclonal, anti-DDX21 (10528-1-AP; Proteintech) rabbit polyclonal, anti-C7orf50 (20797-1-AP; Proteintech) rabbit polyclonal, anti-MYBBP1A (14524-1-AP; Proteintech) rabbit polyclonal, anti-LYRIC (40-6500; Invitrogen) rabbit polyclonal, anti-SND1 (60265-1-Ig; Proteintech) mouse monoclonal, anti-NPM3 (11960-1-AP; Proteintech) rabbit polyclonal, anti-LYAR (PA5-98969; Invitrogen) rabbit polyclonal, anti-MRTO4 (H00051154-B01P; Thermo Fisher Scientific) mouse polyclonal, anti-NOG1 (GTX110826; Genetex) rabbit polyclonal, anti-NOP2 (10448-1-AP; Proteintech) rabbit polyclonal, anti-uL18 (A303-933A; BETHYL) rabbit polyclonal, anti-uS3 (GTX54720; Genetex) rabbit polyclonal, anti-tubulin β (T7816; Sigma-Aldrich) mouse monoclonal, anti-GAPDH (ab8245; Abcam) mouse monoclonal; anti-PES1 and anti-NST (courtesy from E Kremmer); anti-URB1 (PA5-53787; Thermo Fisher Scientific), anti-FBL (ab5821; Abcam), anti-RPA194 (SC-48385; Santa Cruz).

Techniques: Magnetic Beads, Recombinant, Negative Control, SDS Page, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: The RNA component of SRP, 7SL, is synthesized in the nucleoplasm by RNA polymerase III, where its 3′ end is bound by protein La. Other maturation steps occur in the nucleolus and/or putatively the Cajal bodies (CBs) as indicated. Five of the six SRP protein subunits (SRP9, SRP14, SRP19, SRP68, and SRP72) are assembled in the nucleolus (see text for details). After export to the cytoplasm, the sixth subunit (SRP54) is assembled, aided by the SMN complex, and mature SRP is produced. The Alu and S domains of 7SL are indicated.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Synthesized, Produced

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D, E, F, G, H) For U2OS Flp-In T-REx cells (left column), the expression of the genes encoding GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), and GFP-SRP14 (D) was induced by addition of Dox (1 μg/ml) to the medium and incubation for the indicated time. (E, F, G, H) Same concentration of Dox was used to induce the expression of the genes encoding GFP-SRP19 (E), GFP-SRP72 (F), GFP-SRP9 (G), and GFP-SRP14 (H) in HEK293 Flp-In T-REx cells (right column). Amounts of each GFP-SRP protein and the corresponding endogenous protein were analyzed by SDS–PAGE and WB with specific antibodies. GAPDH or tubulin β was used as a loading control. The expression of a given GFP-tagged SRP construct generally led to a reduced level of the corresponding endogenous protein. To us, this suggests that cells somehow regulate overall amounts of SRP proteins via a mechanism that remains to be determined. (I, J) Proteins SRP14 and SRP72 exist in different isoforms. U2OS Flp-In T-REx cells were transfected for 48 h with siRNAs specific to the mRNA encoding the SRP14 protein (si-SRP14) (I) or the SRP72 protein (si-SRP72) (J). Control siRNAs targeting the mRNA encoding luciferase (si-Luc) were used as negative controls. Total extracts were prepared and analyzed by SDS–PAGE and WB. Tubulin β was used as a loading control. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Expressing, Incubation, Concentration Assay, SDS Page, Control, Construct, Transfection, Luciferase, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D) Total extracts were produced from HEK293 Flp-In T-REx cells having expressed for 3 h one of the following proteins: GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), or GFP-SRP14 (D). IPs were carried out, in the presence (+) and absence (−) of RNase A, with GFP-Trap beads and either one of these extracts or an extract of parental HEK293 Flp-In T-REx cells (Control). The immunoprecipitate (IP) and a fraction of the total cell extract (5%) (Total) were analyzed by SDS–PAGE and WB with antibodies against the indicated proteins. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Produced, Control, SDS Page, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D) IP-SILAC analyses performed on HEK293 Flp-In T-REx cells expressing GFP-SRP19 (A), GFP-SRP9 (C), or GFP-SRP14 (D) for 3 h or on U2OS Flp-In T-REx cells expressing GFP-SRP72 for 3 h (B). The graph displays the log 2 of the SILAC ratio (y-axis, specific IP versus control IP performed with parental Flp-In T-REx cells) as a function of signal abundance (x-axis, log 10 (intensity)/MW). Each dot represents a protein. The labeled dots were arbitrarily selected to highlight proteins relevant to this study and families of proteins (see Key below the graphs) associated with GFP-SRP proteins. Analysis of the functions of the associated proteins was performed with the Gene Ontology Resource and UniProt. The full hit list with Significance B values is given in Table S1. The indicated percentage of nucleolar proteins and/or proteins involved in ribosome biogenesis, as well as the one of ribosomal proteins, represents the percentages in the number of these classes of proteins among all the associated proteins with the GFP-SRP protein analyzed and with a SILAC ratio above 1.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Expressing, Control, Labeling

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D) Synthesis of GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), and GFP-SRP14 (D) was induced for 12 h in U2OS Flp-In T-REx cells. Total extracts were prepared, and IPs were carried out with GFP-Trap beads on extracts from parental U2OS Flp-In T-REx cells (Control) and U2OS Flp-In T-REx cells expressing one of the GFP-SRP proteins. The immunoprecipitate (IP) and a fraction (5%) of the total protein extract (Total) were analyzed by SDS–PAGE and WB with antibodies against the indicated proteins. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Control, Expressing, SDS Page, Molecular Weight

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A, B, C, D, E, F) Expression of the gene encoding GFP-SRP19 (A), GFP-SRP72 (B), GFP-SRP9 (C), or GFP-SRP14 (E) in HEK293 Flp-In T-REx cells or GFP-SRP9 (D) or GFP-SRP14 (F) in U2OS Flp-In T-Rex cells was induced with Dox for 12 h. The subcellular localization of the indicated protein was then analyzed by direct fluorescence of the GFP tag. Nuclei were stained in blue with DAPI. Images were acquired with an epifluorescence microscope. GFP-SRP proteins are in green. Scale bar: 15 μm. (G, H) Expression of the genes encoding GFP-SRP19 and GFP-SRP72 was induced with Dox for 12 h in HEK293 Flp-In T-REx cells. Double IF experiments were performed with anti-coilin (a marker of CBs) and anti-NCL (a marker of nucleolus) antibodies. Images were acquired with a confocal microscope. GFP-SRP19 and GFP-SRP72 are in cyan, and their localization was determined by direct GFP fluorescence analysis. Coilin is in magenta, and NCL is in yellow. White arrows indicate the co-localization of the GFP-SRP19 or GFP-SRP72 protein with coilin. Scale bar: 7 μm.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Expressing, Fluorescence, Staining, Microscopy, Marker

Journal: Life Science Alliance

Article Title: The nucleolar phase of signal recognition particle assembly

doi: 10.26508/lsa.202402614

Figure Lengend Snippet: (A) Venn diagram showing the intersection between nucleolar proteins and/or proteins involved in ribosome biogenesis present in the GFP-SRP9, GFP-SRP14, GFP-SRP19, and GFP-SRP72 interactomes as determined by IP-SILAC analysis. The diagram includes all the proteins associated with a SILAC ratio up to 1. The ones associated with at least 3 GFP-SRP proteins are listed in red. (B) IPs were carried out on U2OS Flp-In T-REx cell total extracts, using anti-SRP19 (upper panels) and anti-SRP9 (lower panels) antibodies bound to magnetic beads with recombinant protein A (Dynabeads Protein A). Beads alone were used as a negative control (Control). The immunoprecipitate (IP) and a fraction of the total cell extract (5%) (Total) were analyzed by SDS–PAGE. The indicated proteins were revealed by WB. The molecular weight ladder (MW) loaded in parallel with the samples is indicated.

Article Snippet: Stable HEK293 and U2OS cell lines expressing a gene encoding GFP-tagged SRP9, SRP14, SRP19, or SRP72 under the control of a tetracycline-regulated CMV/TetO 2 promoter were created with the Flp-In T-REx system (Thermo Fisher Scientific) as recommended by the manufacturer, using the HEK293 or U2OS Flp-In T-REx cell lines and the pcDNA5/FRT/TO plasmid encoding the desired ORF.

Techniques: Magnetic Beads, Recombinant, Negative Control, Control, SDS Page, Molecular Weight