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prpsap2  (Proteintech)
93
Proteintech prpsap2
A Phylogenetic distribution of PRPS homologs in eukaryotes, with presence/absence of NHR-containing PRPS homologs in opisthokonts. <t>PRPSAP2</t> denotes orthologs of mammalian PRPSAP2; Prs1 and Prs5 represent S. cerevisiae Prs1 and Prs5, respectively. Black asterisk indicates Class II presence across most metazoans, excluding Craniata. Red asterisks indicate Class II PRPS identified in only two Nuclearia species and one Chytridomycota species. B Conserved splice site junctions among PRPS homologs across different representative organisms in Opisthokonta. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Multiple sequence alignment (MSA) of translated sequences via Clustal Omega highlights a conserved splice site junction (red arrow) with adjacent amino acids shown. Top bar shows full H. sapiens PRPS1 with RF loop (red box) and CF loop (black box). Dotted red and black lines project corresponding RF and CF loops positions, respectively onto other homologs. Insertions in RF and CF loops of PRPSAP2 and Prs5 shown with red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length). C Phylogenetic distribution of PRPS homologs in chordates. Mostly present – indicates present in most taxa. Black asterisk highlights that Class II PRPS is found in most urochordates, except Oikopleura , which has additional Class I PRPS homologs not observed in other organisms from this clade. D , E Conserved splice site junctions between PRPS1 and PRPS2 ( D ), and PRPSAP2 and PRPSAP1 ( E ) across different representative organisms in jawed Vertebrata. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Dotted red and black lines denote RF and CF loop regions, respectively; insertions in PRPSAP2 and PRPSAP1 shown as red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length).
Prpsap2, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti prpsap2  (Proteintech)
93
Proteintech rabbit polyclonal anti prpsap2
A Phylogenetic distribution of PRPS homologs in eukaryotes, with presence/absence of NHR-containing PRPS homologs in opisthokonts. <t>PRPSAP2</t> denotes orthologs of mammalian PRPSAP2; Prs1 and Prs5 represent S. cerevisiae Prs1 and Prs5, respectively. Black asterisk indicates Class II presence across most metazoans, excluding Craniata. Red asterisks indicate Class II PRPS identified in only two Nuclearia species and one Chytridomycota species. B Conserved splice site junctions among PRPS homologs across different representative organisms in Opisthokonta. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Multiple sequence alignment (MSA) of translated sequences via Clustal Omega highlights a conserved splice site junction (red arrow) with adjacent amino acids shown. Top bar shows full H. sapiens PRPS1 with RF loop (red box) and CF loop (black box). Dotted red and black lines project corresponding RF and CF loops positions, respectively onto other homologs. Insertions in RF and CF loops of PRPSAP2 and Prs5 shown with red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length). C Phylogenetic distribution of PRPS homologs in chordates. Mostly present – indicates present in most taxa. Black asterisk highlights that Class II PRPS is found in most urochordates, except Oikopleura , which has additional Class I PRPS homologs not observed in other organisms from this clade. D , E Conserved splice site junctions between PRPS1 and PRPS2 ( D ), and PRPSAP2 and PRPSAP1 ( E ) across different representative organisms in jawed Vertebrata. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Dotted red and black lines denote RF and CF loop regions, respectively; insertions in PRPSAP2 and PRPSAP1 shown as red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length).
Rabbit Polyclonal Anti Prpsap2, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sp2 antibody  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology sp2 antibody
FIGURE 5 | <t>SP2,</t> poorly expressed in OA, represses DVL1 transcription. (A) Transcription factor binding sites near the mouse DVL1 promoter region predicted using the JASPAR Transcription Factors plugin from the UCSC Genome Browser. (B) Intersections of the predicted transcription factors and the significant DEGs obtained from high-throughput sequencing. (C) Positive staining of SP2 in the joint cartilage determined using IHC. (D) SP2 mRNA expression in the joint cartilage determined using RT-qPCR. (E) Binding between SP2 and the DVL1 promoter in chondrocytes determined using ChIP-qPCR assay; the extracted chondrocytes were administered lentiviral vectors carrying OE-NC or OE-SP2. (F) mRNA and (G) protein level of SP2 in mouse chondrocytes determined using RT-qPCR and WB analysis. (H) mRNA expression of DVL1 in mouse chondrocytes analyzed using RT-qPCR. (I) Regulation of SP2 on transcription activity of the DVL1 promoter in chondrocytes determined using the dual lucifer- ase reporter gene assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C–I).
Sp2 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sp2  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology sp2
Figure 4. Transcription of the Dapk3 gene is promoted by <t>Sp2.</t> A) Three potential REs of the Dapk3 gene were predicted by the JASPAR database. B–G) TM3 cells were transfected with 0, 2, and 4 μg pcDNA3.1-Sp2 for 48 h or 250 pmol μL−1 si-Sp2 for 24 h, and the mRNA and protein levels of DAPK3 were determined. H–J) After TM3 cells were exposed to 0 or 400 μm DBP for 24 h in the presence or absence of 250 pmol μL−1 si-Sp2, the mRNA and protein contents of DAPK3 were detected. K) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence or absence of pGL4.2- Dapk3 prom, the luciferase activity was detected. L) TM3 cells were transfected with pGL4.2-RE1, pGL4.2-RE2, or pGL4.2-RE3 together with or without pcDNA3.1-Sp2, the luciferase activity was then determined. M) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence of pGL4.2-RE3 or pGL4.2-mutRE3, the luciferase activity was determined. N) The binding of Sp2 to the RE3 site of the Dapk3 gene was verified by ChIP- qPCR. Data are represented as means ± SEM, n = 3. *P < 0.05, by one-way ANOVA with LSD method (B, D, H, J, K, and M) or independent sample t-test (E, G, L, and N).
Sp2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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l asaccharolyticus dsm106493 strain  (DSMZ)
93
DSMZ l asaccharolyticus dsm106493 strain
Figure 4. Transcription of the Dapk3 gene is promoted by <t>Sp2.</t> A) Three potential REs of the Dapk3 gene were predicted by the JASPAR database. B–G) TM3 cells were transfected with 0, 2, and 4 μg pcDNA3.1-Sp2 for 48 h or 250 pmol μL−1 si-Sp2 for 24 h, and the mRNA and protein levels of DAPK3 were determined. H–J) After TM3 cells were exposed to 0 or 400 μm DBP for 24 h in the presence or absence of 250 pmol μL−1 si-Sp2, the mRNA and protein contents of DAPK3 were detected. K) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence or absence of pGL4.2- Dapk3 prom, the luciferase activity was detected. L) TM3 cells were transfected with pGL4.2-RE1, pGL4.2-RE2, or pGL4.2-RE3 together with or without pcDNA3.1-Sp2, the luciferase activity was then determined. M) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence of pGL4.2-RE3 or pGL4.2-mutRE3, the luciferase activity was determined. N) The binding of Sp2 to the RE3 site of the Dapk3 gene was verified by ChIP- qPCR. Data are represented as means ± SEM, n = 3. *P < 0.05, by one-way ANOVA with LSD method (B, D, H, J, K, and M) or independent sample t-test (E, G, L, and N).
L Asaccharolyticus Dsm106493 Strain, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


A Phylogenetic distribution of PRPS homologs in eukaryotes, with presence/absence of NHR-containing PRPS homologs in opisthokonts. PRPSAP2 denotes orthologs of mammalian PRPSAP2; Prs1 and Prs5 represent S. cerevisiae Prs1 and Prs5, respectively. Black asterisk indicates Class II presence across most metazoans, excluding Craniata. Red asterisks indicate Class II PRPS identified in only two Nuclearia species and one Chytridomycota species. B Conserved splice site junctions among PRPS homologs across different representative organisms in Opisthokonta. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Multiple sequence alignment (MSA) of translated sequences via Clustal Omega highlights a conserved splice site junction (red arrow) with adjacent amino acids shown. Top bar shows full H. sapiens PRPS1 with RF loop (red box) and CF loop (black box). Dotted red and black lines project corresponding RF and CF loops positions, respectively onto other homologs. Insertions in RF and CF loops of PRPSAP2 and Prs5 shown with red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length). C Phylogenetic distribution of PRPS homologs in chordates. Mostly present – indicates present in most taxa. Black asterisk highlights that Class II PRPS is found in most urochordates, except Oikopleura , which has additional Class I PRPS homologs not observed in other organisms from this clade. D , E Conserved splice site junctions between PRPS1 and PRPS2 ( D ), and PRPSAP2 and PRPSAP1 ( E ) across different representative organisms in jawed Vertebrata. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Dotted red and black lines denote RF and CF loop regions, respectively; insertions in PRPSAP2 and PRPSAP1 shown as red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length).

Journal: Nature Communications

Article Title: The role of gene duplication and paralog specialisation in the evolution of the mammalian PRPS complex

doi: 10.1038/s41467-025-61216-z

Figure Lengend Snippet: A Phylogenetic distribution of PRPS homologs in eukaryotes, with presence/absence of NHR-containing PRPS homologs in opisthokonts. PRPSAP2 denotes orthologs of mammalian PRPSAP2; Prs1 and Prs5 represent S. cerevisiae Prs1 and Prs5, respectively. Black asterisk indicates Class II presence across most metazoans, excluding Craniata. Red asterisks indicate Class II PRPS identified in only two Nuclearia species and one Chytridomycota species. B Conserved splice site junctions among PRPS homologs across different representative organisms in Opisthokonta. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Multiple sequence alignment (MSA) of translated sequences via Clustal Omega highlights a conserved splice site junction (red arrow) with adjacent amino acids shown. Top bar shows full H. sapiens PRPS1 with RF loop (red box) and CF loop (black box). Dotted red and black lines project corresponding RF and CF loops positions, respectively onto other homologs. Insertions in RF and CF loops of PRPSAP2 and Prs5 shown with red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length). C Phylogenetic distribution of PRPS homologs in chordates. Mostly present – indicates present in most taxa. Black asterisk highlights that Class II PRPS is found in most urochordates, except Oikopleura , which has additional Class I PRPS homologs not observed in other organisms from this clade. D , E Conserved splice site junctions between PRPS1 and PRPS2 ( D ), and PRPSAP2 and PRPSAP1 ( E ) across different representative organisms in jawed Vertebrata. Gene structures for PRPS, PRPSAP2, and Prs5-encoding genes shown with exons as colored boxes; introns not displayed. Dotted red and black lines denote RF and CF loop regions, respectively; insertions in PRPSAP2 and PRPSAP1 shown as red and black hatch marks, respectively (hatch marks not to scale; NHRs vary in length).

Article Snippet: After blocking, the membranes were washed and incubated overnight at 4 °C with primary antibodies (diluted 1:1000) prepared in 3% BSA in TBS-T. Primary antibodies used were: CAD (Cell Signaling #93925), TCP1-η (Santa Cruz #sc-271951), FASN (Cell Signaling #3180), FLC (Santa Cruz #sc-390558), HK2 (Cell Signaling #2867), AK2 (Santa Cruz #sc-374095), PRPS1/2 (Santa Cruz #sc-100822), PRPS1 (Proteintech #15549-1-AP), PRPS2 (Sigma #SAB2107995), PRPS1/2/3 (Santa Cruz #sc-376440), PRPSAP1 (Santa Cruz #sc-398422), PRPSAP2 (Proteintech #17814-1-AP), HSP90 (Cell Signaling #4877), β-Actin (Cell Signaling #4970; Cell Signaling #3700), ALFA-HRP (SynapticSystems # N1505-HRP), XO (Abcam #109235), Ras (G12V Mutant Specific) (Cell Signaling #14412), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling #4376), p44/42 MAPK (Erk1/2) (Cell Signaling #9102), β-Tubulin (Cell Signaling #2128), Phospho-AMPKα (Thr172) (Cell Signaling #2535), AMPKα (Cell Signaling #2532), cleaved PARP1 (Abcam #32064), GAPDH (Cell Signaling #5174), HPRT (Abcam #109021).

Techniques: Sequencing

A Structure of dimeric human PRPS1 (PDB: 2HCR), with a zoom-in of catalytic site highlighting metal binding site (Cd 2+ ), AMP (represents AMP moiety of ATP), SO 4 2− (represents 5′-phosphate of R5P), and several conserved active site residues (magenta). D171 coordinates metal binding, K194 interacts with ATP, R196, and T225 interact with R5P, and N200 stabilizes the catalytic loop. B WebLogo depicting MSA of active and regulatory site residues from PRPS1 and PRPSAP2 from representative organisms in opisthokonts ( n = 44 each). Numbers below indicate corresponding residue positions in human PRPS1 ( NP_002755.1 ) and PRPSAP2 ( NP_001340030.1 ). Asterisks denote residues conserved in PRPS but substituted in PRPSAP2 (also shown in A ). C AlphaFold2-predicted structure of human PRPSAP2 ( NP_001340030.1 ), with a zoom-in highlighting four non-conserved residues (magenta) at corresponding active site positions in PRPS1 shown in ( A ). AMP modeled to indicate putative ATP binding site. D Trimeric structure of human PRPS1 (PDB: 2HCR). Red and magenta residues in dashed box represent dimer interface residues in bent ( B , C ) and parallel ( A , B ) dimers, respectively. Amino acid sequence of B. subtilis PRPS aligned with Opisthokonta PRPS homologs to identify corresponding dimer interface residues for WebLogo. Representative sequences include PRPS1 ( n = 44), PRPSAP2 ( n = 44) from opisthokonts, and PRPS2 ( n = 46), PRPSAP1 ( n = 92) from jawed vertebrates. Residue numbers based on human PRPS1 ( NP_002755.1 ), PRPS2 ( NP_002756.1 ), PRPSAP1 ( AAH09012.1 ), and PRPSAP2 ( NP_001340030.1 ). E PRPS paralogs from S. cerevisiae , H. sapiens, R. potamoides, T. trahens, and D. rotans showing relative NHR positions in paralogs with expanded CF and/or RF loops. Open bar for each representative species represents full polypeptide sequences of their ancestral PRPS (bold). Amino acid positions corresponding to RF and CF loops labeled within bars. NHR insertions sites for Prs1, Prs5, PRPSAP1, PRPSAP2, PrsB, and PRPSAP-like homologs marked by triangles above/below bars. Numbers next to triangles indicate amino acid count per NHR.

Journal: Nature Communications

Article Title: The role of gene duplication and paralog specialisation in the evolution of the mammalian PRPS complex

doi: 10.1038/s41467-025-61216-z

Figure Lengend Snippet: A Structure of dimeric human PRPS1 (PDB: 2HCR), with a zoom-in of catalytic site highlighting metal binding site (Cd 2+ ), AMP (represents AMP moiety of ATP), SO 4 2− (represents 5′-phosphate of R5P), and several conserved active site residues (magenta). D171 coordinates metal binding, K194 interacts with ATP, R196, and T225 interact with R5P, and N200 stabilizes the catalytic loop. B WebLogo depicting MSA of active and regulatory site residues from PRPS1 and PRPSAP2 from representative organisms in opisthokonts ( n = 44 each). Numbers below indicate corresponding residue positions in human PRPS1 ( NP_002755.1 ) and PRPSAP2 ( NP_001340030.1 ). Asterisks denote residues conserved in PRPS but substituted in PRPSAP2 (also shown in A ). C AlphaFold2-predicted structure of human PRPSAP2 ( NP_001340030.1 ), with a zoom-in highlighting four non-conserved residues (magenta) at corresponding active site positions in PRPS1 shown in ( A ). AMP modeled to indicate putative ATP binding site. D Trimeric structure of human PRPS1 (PDB: 2HCR). Red and magenta residues in dashed box represent dimer interface residues in bent ( B , C ) and parallel ( A , B ) dimers, respectively. Amino acid sequence of B. subtilis PRPS aligned with Opisthokonta PRPS homologs to identify corresponding dimer interface residues for WebLogo. Representative sequences include PRPS1 ( n = 44), PRPSAP2 ( n = 44) from opisthokonts, and PRPS2 ( n = 46), PRPSAP1 ( n = 92) from jawed vertebrates. Residue numbers based on human PRPS1 ( NP_002755.1 ), PRPS2 ( NP_002756.1 ), PRPSAP1 ( AAH09012.1 ), and PRPSAP2 ( NP_001340030.1 ). E PRPS paralogs from S. cerevisiae , H. sapiens, R. potamoides, T. trahens, and D. rotans showing relative NHR positions in paralogs with expanded CF and/or RF loops. Open bar for each representative species represents full polypeptide sequences of their ancestral PRPS (bold). Amino acid positions corresponding to RF and CF loops labeled within bars. NHR insertions sites for Prs1, Prs5, PRPSAP1, PRPSAP2, PrsB, and PRPSAP-like homologs marked by triangles above/below bars. Numbers next to triangles indicate amino acid count per NHR.

Article Snippet: After blocking, the membranes were washed and incubated overnight at 4 °C with primary antibodies (diluted 1:1000) prepared in 3% BSA in TBS-T. Primary antibodies used were: CAD (Cell Signaling #93925), TCP1-η (Santa Cruz #sc-271951), FASN (Cell Signaling #3180), FLC (Santa Cruz #sc-390558), HK2 (Cell Signaling #2867), AK2 (Santa Cruz #sc-374095), PRPS1/2 (Santa Cruz #sc-100822), PRPS1 (Proteintech #15549-1-AP), PRPS2 (Sigma #SAB2107995), PRPS1/2/3 (Santa Cruz #sc-376440), PRPSAP1 (Santa Cruz #sc-398422), PRPSAP2 (Proteintech #17814-1-AP), HSP90 (Cell Signaling #4877), β-Actin (Cell Signaling #4970; Cell Signaling #3700), ALFA-HRP (SynapticSystems # N1505-HRP), XO (Abcam #109235), Ras (G12V Mutant Specific) (Cell Signaling #14412), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling #4376), p44/42 MAPK (Erk1/2) (Cell Signaling #9102), β-Tubulin (Cell Signaling #2128), Phospho-AMPKα (Thr172) (Cell Signaling #2535), AMPKα (Cell Signaling #2532), cleaved PARP1 (Abcam #32064), GAPDH (Cell Signaling #5174), HPRT (Abcam #109021).

Techniques: Binding Assay, Residue, Sequencing, Labeling

A–C Western blot analysis of SEC fractions collected from native whole-cell lysates of NIH3T3 AP1 KO cells stably expressing AP1 lacking the non-homologous region (NHR) ( A ), AP1/AP2 KO cells stably expressing chimeric AP1 with AP2’s N-terminus (residues 1–95) B and chimeric AP2 with AP1’s N-terminus (residues 1–95) ( C ). D Schematic representation of alternative start sites in mammalian AP1 and AP2 and their consequent translation into short and long isoforms. Base positions correspond to the mouse homologs. TSS1 and TSS2 represent upstream and downstream translation start sites, respectively. E Multiple isoforms for AP1 and AP2 detected under optimal SDS-PAGE resolution. Arrows in immunoblots indicate the longer isoforms of AP1 and AP2, corresponding to N-terminal leader sequences of 29 and 12 amino acids, respectively. F Phylogenetic distribution of PRPS homologs (PRPS1, PRPS2, PRPSAP2, PRPSAP2 with N-terminal leader sequence, PRPSAP1, and PRPSAP1 with N-terminal leader sequence) in chordates. Presence/absence noted across the tree. PRPSAP2 and PRPSAP1 isoforms with N-terminal leader sequences emerged in ancestors of Amniota and Osteichthyes, respectively. G–J Western blot analysis of SEC fractions collected from NIH3T3 AP1/AP2 KO cells stably expressing the short isoform of AP1 ( G ), long isoform of AP1 ( H ), short isoform of AP2 ( I ), and long isoform of AP2 ( J ). Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. Circular pictograms below SEC immunoblots schematize PRPS complex configurations. Double circle denotes multiple copies of the protein interacting in a heteromeric complex. Double circle with dotted inner circle denotes multiple copies forming homo-oligomers. Single circle denotes a single protein interacting within the complex. Circle with inner vertical lines denotes proteins forming a trimer or tetramer. Western blot data ( A – C , E , G – J ) are representative of at least 2 biological repeats. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: The role of gene duplication and paralog specialisation in the evolution of the mammalian PRPS complex

doi: 10.1038/s41467-025-61216-z

Figure Lengend Snippet: A–C Western blot analysis of SEC fractions collected from native whole-cell lysates of NIH3T3 AP1 KO cells stably expressing AP1 lacking the non-homologous region (NHR) ( A ), AP1/AP2 KO cells stably expressing chimeric AP1 with AP2’s N-terminus (residues 1–95) B and chimeric AP2 with AP1’s N-terminus (residues 1–95) ( C ). D Schematic representation of alternative start sites in mammalian AP1 and AP2 and their consequent translation into short and long isoforms. Base positions correspond to the mouse homologs. TSS1 and TSS2 represent upstream and downstream translation start sites, respectively. E Multiple isoforms for AP1 and AP2 detected under optimal SDS-PAGE resolution. Arrows in immunoblots indicate the longer isoforms of AP1 and AP2, corresponding to N-terminal leader sequences of 29 and 12 amino acids, respectively. F Phylogenetic distribution of PRPS homologs (PRPS1, PRPS2, PRPSAP2, PRPSAP2 with N-terminal leader sequence, PRPSAP1, and PRPSAP1 with N-terminal leader sequence) in chordates. Presence/absence noted across the tree. PRPSAP2 and PRPSAP1 isoforms with N-terminal leader sequences emerged in ancestors of Amniota and Osteichthyes, respectively. G–J Western blot analysis of SEC fractions collected from NIH3T3 AP1/AP2 KO cells stably expressing the short isoform of AP1 ( G ), long isoform of AP1 ( H ), short isoform of AP2 ( I ), and long isoform of AP2 ( J ). Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. Circular pictograms below SEC immunoblots schematize PRPS complex configurations. Double circle denotes multiple copies of the protein interacting in a heteromeric complex. Double circle with dotted inner circle denotes multiple copies forming homo-oligomers. Single circle denotes a single protein interacting within the complex. Circle with inner vertical lines denotes proteins forming a trimer or tetramer. Western blot data ( A – C , E , G – J ) are representative of at least 2 biological repeats. Source data are provided as a Source Data file.

Article Snippet: After blocking, the membranes were washed and incubated overnight at 4 °C with primary antibodies (diluted 1:1000) prepared in 3% BSA in TBS-T. Primary antibodies used were: CAD (Cell Signaling #93925), TCP1-η (Santa Cruz #sc-271951), FASN (Cell Signaling #3180), FLC (Santa Cruz #sc-390558), HK2 (Cell Signaling #2867), AK2 (Santa Cruz #sc-374095), PRPS1/2 (Santa Cruz #sc-100822), PRPS1 (Proteintech #15549-1-AP), PRPS2 (Sigma #SAB2107995), PRPS1/2/3 (Santa Cruz #sc-376440), PRPSAP1 (Santa Cruz #sc-398422), PRPSAP2 (Proteintech #17814-1-AP), HSP90 (Cell Signaling #4877), β-Actin (Cell Signaling #4970; Cell Signaling #3700), ALFA-HRP (SynapticSystems # N1505-HRP), XO (Abcam #109235), Ras (G12V Mutant Specific) (Cell Signaling #14412), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling #4376), p44/42 MAPK (Erk1/2) (Cell Signaling #9102), β-Tubulin (Cell Signaling #2128), Phospho-AMPKα (Thr172) (Cell Signaling #2535), AMPKα (Cell Signaling #2532), cleaved PARP1 (Abcam #32064), GAPDH (Cell Signaling #5174), HPRT (Abcam #109021).

Techniques: Western Blot, Stable Transfection, Expressing, SDS Page, Sequencing

FIGURE 5 | SP2, poorly expressed in OA, represses DVL1 transcription. (A) Transcription factor binding sites near the mouse DVL1 promoter region predicted using the JASPAR Transcription Factors plugin from the UCSC Genome Browser. (B) Intersections of the predicted transcription factors and the significant DEGs obtained from high-throughput sequencing. (C) Positive staining of SP2 in the joint cartilage determined using IHC. (D) SP2 mRNA expression in the joint cartilage determined using RT-qPCR. (E) Binding between SP2 and the DVL1 promoter in chondrocytes determined using ChIP-qPCR assay; the extracted chondrocytes were administered lentiviral vectors carrying OE-NC or OE-SP2. (F) mRNA and (G) protein level of SP2 in mouse chondrocytes determined using RT-qPCR and WB analysis. (H) mRNA expression of DVL1 in mouse chondrocytes analyzed using RT-qPCR. (I) Regulation of SP2 on transcription activity of the DVL1 promoter in chondrocytes determined using the dual lucifer- ase reporter gene assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C–I).

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 5 | SP2, poorly expressed in OA, represses DVL1 transcription. (A) Transcription factor binding sites near the mouse DVL1 promoter region predicted using the JASPAR Transcription Factors plugin from the UCSC Genome Browser. (B) Intersections of the predicted transcription factors and the significant DEGs obtained from high-throughput sequencing. (C) Positive staining of SP2 in the joint cartilage determined using IHC. (D) SP2 mRNA expression in the joint cartilage determined using RT-qPCR. (E) Binding between SP2 and the DVL1 promoter in chondrocytes determined using ChIP-qPCR assay; the extracted chondrocytes were administered lentiviral vectors carrying OE-NC or OE-SP2. (F) mRNA and (G) protein level of SP2 in mouse chondrocytes determined using RT-qPCR and WB analysis. (H) mRNA expression of DVL1 in mouse chondrocytes analyzed using RT-qPCR. (I) Regulation of SP2 on transcription activity of the DVL1 promoter in chondrocytes determined using the dual lucifer- ase reporter gene assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C–I).

Article Snippet: The samples were then incubated with SP2 antibody (1:1000, sc- 166575, Santa Cruz Biotechnology Inc., Santa Cruz, California, United States) and mouse IgG isotype control (1:100, NBP1- 97142, Novus Biologicals, Littleton, Colorado, United States) overnight at 4°C.

Techniques: Binding Assay, Next-Generation Sequencing, Staining, Expressing, Quantitative RT-PCR, ChIP-qPCR, Activity Assay, Reporter Gene Assay

FIGURE 6 | DVL1 upregulation aggravates cartilage injury in mice alleviated by SP2. DMM-challenged mice were introduced with adenoviral vectors carrying OE-NC/OE-SP2 alone or with the additional OE-NC/OE-DVL1. (A) mRNA expression of SP2 and DVL1 in the joint cartilage deter- mined using RT-qPCR. (B) Positive staining of SP2 and DVL1 in the joint cartilage determined using IHC. (C) Protein levels of β-catenin in the joint cartilage determined using WB analysis. (D) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (E) Positive TRAP staining of the mouse knee joints. (F) Protein levels of COL10A1 and MMP13 in the mouse cartilage determined using WB analysis. Each group contained five mice. Differences were compared by ANOVA (A–F).

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 6 | DVL1 upregulation aggravates cartilage injury in mice alleviated by SP2. DMM-challenged mice were introduced with adenoviral vectors carrying OE-NC/OE-SP2 alone or with the additional OE-NC/OE-DVL1. (A) mRNA expression of SP2 and DVL1 in the joint cartilage deter- mined using RT-qPCR. (B) Positive staining of SP2 and DVL1 in the joint cartilage determined using IHC. (C) Protein levels of β-catenin in the joint cartilage determined using WB analysis. (D) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (E) Positive TRAP staining of the mouse knee joints. (F) Protein levels of COL10A1 and MMP13 in the mouse cartilage determined using WB analysis. Each group contained five mice. Differences were compared by ANOVA (A–F).

Article Snippet: The samples were then incubated with SP2 antibody (1:1000, sc- 166575, Santa Cruz Biotechnology Inc., Santa Cruz, California, United States) and mouse IgG isotype control (1:100, NBP1- 97142, Novus Biologicals, Littleton, Colorado, United States) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Staining

FIGURE 7 | DVL1 overexpression increases chondrocyte loss suppressed by SP2. Chondrocytes were administered lentiviral vectors encapsu- lating OE-NC/OE-SP2 or the additional OE-NC/OE-DVL1. (A) mRNA expression of DVL1 in cells determined using RT-qPCR. (B) Apoptosis in cells determined using TUNEL assay. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) Transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Protein levels of SOX9 and COL2A1 in the chondrocytes determined using WB analysis. Three biological replicates were performed. Differences were compared by unpaired t-test (A) or ANOVA (B–E).

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 7 | DVL1 overexpression increases chondrocyte loss suppressed by SP2. Chondrocytes were administered lentiviral vectors encapsu- lating OE-NC/OE-SP2 or the additional OE-NC/OE-DVL1. (A) mRNA expression of DVL1 in cells determined using RT-qPCR. (B) Apoptosis in cells determined using TUNEL assay. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) Transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Protein levels of SOX9 and COL2A1 in the chondrocytes determined using WB analysis. Three biological replicates were performed. Differences were compared by unpaired t-test (A) or ANOVA (B–E).

Article Snippet: The samples were then incubated with SP2 antibody (1:1000, sc- 166575, Santa Cruz Biotechnology Inc., Santa Cruz, California, United States) and mouse IgG isotype control (1:100, NBP1- 97142, Novus Biologicals, Littleton, Colorado, United States) overnight at 4°C.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, TUNEL Assay, Activity Assay

FIGURE 8 | Schematic illustration of the mechanism. In cartilage tissues of osteoarthritic mice, decreased expression of SP2 resulted in its attenuated transcriptional repression of DVL1. Aberrantly expressed DVL1 activated the β-catenin signaling, leading to chondrocyte damage and osteoarthritis progression.

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 8 | Schematic illustration of the mechanism. In cartilage tissues of osteoarthritic mice, decreased expression of SP2 resulted in its attenuated transcriptional repression of DVL1. Aberrantly expressed DVL1 activated the β-catenin signaling, leading to chondrocyte damage and osteoarthritis progression.

Article Snippet: The samples were then incubated with SP2 antibody (1:1000, sc- 166575, Santa Cruz Biotechnology Inc., Santa Cruz, California, United States) and mouse IgG isotype control (1:100, NBP1- 97142, Novus Biologicals, Littleton, Colorado, United States) overnight at 4°C.

Techniques: Expressing

Figure 4. Transcription of the Dapk3 gene is promoted by Sp2. A) Three potential REs of the Dapk3 gene were predicted by the JASPAR database. B–G) TM3 cells were transfected with 0, 2, and 4 μg pcDNA3.1-Sp2 for 48 h or 250 pmol μL−1 si-Sp2 for 24 h, and the mRNA and protein levels of DAPK3 were determined. H–J) After TM3 cells were exposed to 0 or 400 μm DBP for 24 h in the presence or absence of 250 pmol μL−1 si-Sp2, the mRNA and protein contents of DAPK3 were detected. K) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence or absence of pGL4.2- Dapk3 prom, the luciferase activity was detected. L) TM3 cells were transfected with pGL4.2-RE1, pGL4.2-RE2, or pGL4.2-RE3 together with or without pcDNA3.1-Sp2, the luciferase activity was then determined. M) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence of pGL4.2-RE3 or pGL4.2-mutRE3, the luciferase activity was determined. N) The binding of Sp2 to the RE3 site of the Dapk3 gene was verified by ChIP- qPCR. Data are represented as means ± SEM, n = 3. *P < 0.05, by one-way ANOVA with LSD method (B, D, H, J, K, and M) or independent sample t-test (E, G, L, and N).

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: DAPK3 is Essential for DBP-Induced Autophagy of Mouse Leydig Cells.

doi: 10.1002/advs.202413936

Figure Lengend Snippet: Figure 4. Transcription of the Dapk3 gene is promoted by Sp2. A) Three potential REs of the Dapk3 gene were predicted by the JASPAR database. B–G) TM3 cells were transfected with 0, 2, and 4 μg pcDNA3.1-Sp2 for 48 h or 250 pmol μL−1 si-Sp2 for 24 h, and the mRNA and protein levels of DAPK3 were determined. H–J) After TM3 cells were exposed to 0 or 400 μm DBP for 24 h in the presence or absence of 250 pmol μL−1 si-Sp2, the mRNA and protein contents of DAPK3 were detected. K) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence or absence of pGL4.2- Dapk3 prom, the luciferase activity was detected. L) TM3 cells were transfected with pGL4.2-RE1, pGL4.2-RE2, or pGL4.2-RE3 together with or without pcDNA3.1-Sp2, the luciferase activity was then determined. M) After TM3 cells were transfected with pcDNA3.1 or pcDNA3.1-Sp2 in the presence of pGL4.2-RE3 or pGL4.2-mutRE3, the luciferase activity was determined. N) The binding of Sp2 to the RE3 site of the Dapk3 gene was verified by ChIP- qPCR. Data are represented as means ± SEM, n = 3. *P < 0.05, by one-way ANOVA with LSD method (B, D, H, J, K, and M) or independent sample t-test (E, G, L, and N).

Article Snippet: The antibodies applied for Western blot (WB), immunohistochemistry (IHC), chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (CoIP) were commercially purchased as follows: Sp2 (Santa Cruz Biotechnology, sc-17814, 1:50 for WB), Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Sc-2003), DAPK3 (Abmart, PK16357, 1:1000 for WB, 1:100 for IHC), Microtubule-associated protein 1 light chain 3 (LC3, Cell signaling technology, #4108, 1:1000 for WB), Autophagy-related gene 5 (Atg 5, Cell signaling technology, #12 994, 1:1000 for WB), β-actin (Cell signaling technology, #4970, 1:1000 for WB), Beclin 1 (Cell signaling technology, #3495, 1:1000 for WB), Mouse IgG antibody (Proteintech, B900620), Rabbit IgG (Proteintech, B900610), LC3B (Proteintech, 18725-1-AP, 1:1600 for IHC), PRKN (Proteintech, 14060-1-AP, 1:1000 for WB), Ub (Proteintech, 10201-2-AP, 1:1000 for WB), HA (Proteintech, 51064-2-AP, 1:4000 for WB), Myc (Proteintech, 60003-2-Ig, 1:5000 for WB), Flag (Affinity, T0053, 1:1000 for WB) and Sp2 (Affinity, DF8721, 1:800 for IHC).

Techniques: Transfection, Luciferase, Activity Assay, Binding Assay, ChIP-qPCR

Figure 5. Sp2 is involved in DBP-induced autophagy of mouse Leydig cells. A–F) The protein levels of LC3, Beclin 1, and Atg 5 and the amount of autophagic vesicles were determined after TM3 cells were transfected with 0, 2, and 4 μg pcDNA3.1-Sp2 for 48 h or 250 pmol μL−1 si-Sp2 for 24 h, respectively. G–I) The protein levels of LC3, Beclin 1, and Atg 5 and the amount of autophagic vesicles were detected after TM3 cells were exposed to 0 or 400 μm DBP with or without Sp2 knockdown. Data are represented as means ± SEM, n = 3. *P < 0.05, by one-way ANOVA with LSD method (B, H) or independent sample t-test (E).

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: DAPK3 is Essential for DBP-Induced Autophagy of Mouse Leydig Cells.

doi: 10.1002/advs.202413936

Figure Lengend Snippet: Figure 5. Sp2 is involved in DBP-induced autophagy of mouse Leydig cells. A–F) The protein levels of LC3, Beclin 1, and Atg 5 and the amount of autophagic vesicles were determined after TM3 cells were transfected with 0, 2, and 4 μg pcDNA3.1-Sp2 for 48 h or 250 pmol μL−1 si-Sp2 for 24 h, respectively. G–I) The protein levels of LC3, Beclin 1, and Atg 5 and the amount of autophagic vesicles were detected after TM3 cells were exposed to 0 or 400 μm DBP with or without Sp2 knockdown. Data are represented as means ± SEM, n = 3. *P < 0.05, by one-way ANOVA with LSD method (B, H) or independent sample t-test (E).

Article Snippet: The antibodies applied for Western blot (WB), immunohistochemistry (IHC), chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (CoIP) were commercially purchased as follows: Sp2 (Santa Cruz Biotechnology, sc-17814, 1:50 for WB), Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Sc-2003), DAPK3 (Abmart, PK16357, 1:1000 for WB, 1:100 for IHC), Microtubule-associated protein 1 light chain 3 (LC3, Cell signaling technology, #4108, 1:1000 for WB), Autophagy-related gene 5 (Atg 5, Cell signaling technology, #12 994, 1:1000 for WB), β-actin (Cell signaling technology, #4970, 1:1000 for WB), Beclin 1 (Cell signaling technology, #3495, 1:1000 for WB), Mouse IgG antibody (Proteintech, B900620), Rabbit IgG (Proteintech, B900610), LC3B (Proteintech, 18725-1-AP, 1:1600 for IHC), PRKN (Proteintech, 14060-1-AP, 1:1000 for WB), Ub (Proteintech, 10201-2-AP, 1:1000 for WB), HA (Proteintech, 51064-2-AP, 1:4000 for WB), Myc (Proteintech, 60003-2-Ig, 1:5000 for WB), Flag (Affinity, T0053, 1:1000 for WB) and Sp2 (Affinity, DF8721, 1:800 for IHC).

Techniques: Transfection, Knockdown

Figure 9. Melatonin attenuates DBP-induced autophagy of mouse Leydig cells through regulating Sp2/DAPK3 and PRKN/DAPK3 signaling pathways. A,B) TM3 cells were treated with 0, 100, 200, and 400 μm H2O2 for 12 h, the expression of Sp2, PRKN, DAPK3, and autophagy-related proteins were detected by Western blot. n = 3. C–E) TM3 cells were exposed to 0 or 400 μm DBP for 24 h in the presence or absence of 500 μm NAC, the protein levels of Sp2, PRKN, DAPK3, and autophagy-related proteins, and the amount of autophagic vesicles were determined by Western blot and TEM, respectively. n = 3. F,G) After male mice were administrated with 0 or 500 mg kg−1 DBP in the presence or absence of 10 mg kg−1 melatonin for 28 d, Western

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: DAPK3 is Essential for DBP-Induced Autophagy of Mouse Leydig Cells.

doi: 10.1002/advs.202413936

Figure Lengend Snippet: Figure 9. Melatonin attenuates DBP-induced autophagy of mouse Leydig cells through regulating Sp2/DAPK3 and PRKN/DAPK3 signaling pathways. A,B) TM3 cells were treated with 0, 100, 200, and 400 μm H2O2 for 12 h, the expression of Sp2, PRKN, DAPK3, and autophagy-related proteins were detected by Western blot. n = 3. C–E) TM3 cells were exposed to 0 or 400 μm DBP for 24 h in the presence or absence of 500 μm NAC, the protein levels of Sp2, PRKN, DAPK3, and autophagy-related proteins, and the amount of autophagic vesicles were determined by Western blot and TEM, respectively. n = 3. F,G) After male mice were administrated with 0 or 500 mg kg−1 DBP in the presence or absence of 10 mg kg−1 melatonin for 28 d, Western

Article Snippet: The antibodies applied for Western blot (WB), immunohistochemistry (IHC), chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (CoIP) were commercially purchased as follows: Sp2 (Santa Cruz Biotechnology, sc-17814, 1:50 for WB), Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Sc-2003), DAPK3 (Abmart, PK16357, 1:1000 for WB, 1:100 for IHC), Microtubule-associated protein 1 light chain 3 (LC3, Cell signaling technology, #4108, 1:1000 for WB), Autophagy-related gene 5 (Atg 5, Cell signaling technology, #12 994, 1:1000 for WB), β-actin (Cell signaling technology, #4970, 1:1000 for WB), Beclin 1 (Cell signaling technology, #3495, 1:1000 for WB), Mouse IgG antibody (Proteintech, B900620), Rabbit IgG (Proteintech, B900610), LC3B (Proteintech, 18725-1-AP, 1:1600 for IHC), PRKN (Proteintech, 14060-1-AP, 1:1000 for WB), Ub (Proteintech, 10201-2-AP, 1:1000 for WB), HA (Proteintech, 51064-2-AP, 1:4000 for WB), Myc (Proteintech, 60003-2-Ig, 1:5000 for WB), Flag (Affinity, T0053, 1:1000 for WB) and Sp2 (Affinity, DF8721, 1:800 for IHC).

Techniques: Protein-Protein interactions, Expressing, Western Blot