Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: List of the antibodies used in this study

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques: Incubation

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: List of the primers used in this study

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques: Sequencing

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: Overview of the CNKSR2 interactome identified in this study. (A) CNKSR2 interactors identified in the DOC and Triton lysates. (B) Molecular function analysis of the CNKSR2 interactors. (C, D) Pathway and process enrichment analysis of the CNKSR2 interactors identified in the DOC (C) and Triton (D) lysates. The color coding of the protein networks corresponds to that of the biological processes in C and D. CNKSR2: Connector enhancer of kinase suppressor of Ras 2; DOC: sodium deoxycholate.

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques:

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: Association of the CNKSR2 interactome with the centrosome. (A, B) Organelle enrichment analysis of the CNKSR2 interactors identified in DOC (A) and Triton (B) lysates. (C) IP-WB assays to verify the interactions of CNKSR2 with CEP290 and DYNC1H1 in the DOC lysates of adult mice prefrontal cortex. (D) Coimmunostaining of CNKSR2 (green, CoraLite647) and CEP63 (red, Alexa Fluor 546) in N2A cells. DAPI was used to stain the nucleus (blue). Arrows indicate the colocalization of CNKSR2 and CEP63. Scale bars: 10 µm. CEP290: Centrosomal protein of 290 kDa; CEP63: centrosomal protein of 63 kDa; CNKSR2: connector enhancer of kinase suppressor of Ras 2; DAPI: 4′,6-diamidino-2-phenylindole; DOC: sodium deoxycholate; DYNC1H1: dynein cytoplasmic 1 heavy chain 1; IF: immunofluorescence; IP: immunoprecipitation; N2A: Neuro 2A; WB: western blot.

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques: Staining, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: Analysis of the sub-centrosome distribution of the CNKSR2 interactors. (A, B) The chord diagrams illustrated the presence of CNKSR2 interactors identified in DOC (A) and Triton (B) lysates in the sub-centrosomal marker interactomes. The identified CNKSR2 interactors were overlaid on published sub-centrosomal marker interactomes (O’Neill et al., 2022), and chords were drawn between the protein and the corresponding sub-centrosomal marker interactomes. The color coding is the same for A and B. CDK5RAP2: CDK5 regulatory subunit associated protein 2; CEP135: centrosomal protein of 135 kDa; CEP152: centrosomal protein of 152 kDa; CEP170: centrosomal protein of 170 kDa; CEP192: centrosomal protein of 192 kDa; CEP63: centrosomal protein of 63 kDa; CNKSR2: Connector Enhancer of Kinase Suppressor of Ras 2; CNTROB: Centrobin; CP110: centriolar coiled-coil protein 110; DOC: sodium deoxycholate; ODF2: outer dense fiber of sperm tails 2; POC5: protein of centriole 5.

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques: Marker

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: CNKSR2 knockdown disturbs centrosome-related functions. (A) Western blot analysis of CNKSR2 expression in N2A cells infected with CNKSR2 KD and NC lentivirus ( n = 3). (B) Typical IF images of N2A cells showed that cells in the CNKSR2 KD group were smaller and rounder than those in the NC group. DAPI was used to stain nuclei (blue). EGFP proteins (green) were expressed by the lentivirus-infected cells. Immunostaining of α-tubulin (red, Alexa Fluor 546) in N2A cells. Scale bars: 10 µm. (C, D) Statistical analysis of the average cell spreading area (C) and cell aspect ratio (D) of N2A cells infected with the lentiviruses ( n = 11 images from three biological replicates; the total number of cells quantified was 268 in the CNKSR2 KD group and 151 in the NC group). (E) CCK8 analysis of N2A cells infected with the lentiviruses ( n = 3). (F) RT-PCR analysis of MKI67 gene expression in N2A cells infected with the lentiviruses ( n = 6). (G) Typical images of the Transwell assays showed that N2A cells in the CNKSR2 KD group had higher motility than those in the NC group ( n = 10). Scale bars: 100 µm. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired two-tailed Student’s t -test). CCK8: cell counting kit-8; CNKSR2: connector enhancer of kinase suppressor of Ras 2; DAPI: 4′,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase IF: immunofluorescence; KD: knock down; MKI67: proliferation marker protein Ki-67; N2A: Neuro 2A; NC: negative control; ns: no significance; RT-PCR: real-time polymerase chain reaction.

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques: Knockdown, Western Blot, Expressing, Infection, Staining, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Cell Counting, Immunofluorescence, Marker, Negative Control, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: CNKSR2 knockdown affects centrosome/microtubule gene expression. RT-PCR analysis of the expression of the centrosomal and microtubule-related genes of N2A cells infected with the CNKSR2 KD and NC lentiviruses. Data are presented as mean ± SD ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired two-tailed Student’s t -test). AKAP9: A-kinase anchor protein 9; CDK5RAP2: CDK5 regulatory subunit associated protein 2; CENPE: centromere-associated protein E; CEP135: centrosomal protein of 135 kDa; CEP152: centrosomal protein of 152 kDa; CEP170: centrosomal protein of 170 kDa; CEP192: centrosomal protein of 192 kDa; CEP290: centrosomal protein of 290 kDa; CEP350: centrosomal protein of 350 kDa; CEP63: centrosomal protein of 63 kDa; CNKSR2: Connector Enhancer of Kinase Suppressor of Ras 2; CNTROB: Centrobin; CP110: centriolar coiled-coil protein 110; DYNC1H1: dynein cytoplasmic 1 heavy chain 1; DYNC2H1: dynein cytoplasmic 2 heavy chain 1; KD: knock down; N2A: Neuro 2A; NC: negative control; ns: no significance; ODF2: outer dense fiber of sperm tails 2; PCNT: Pericentrin; POC5: protein of centriole 5; RT-PCR: real-time polymerase chain reaction.

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Two Tailed Test, Negative Control, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

doi: 10.4103/NRR.NRR-D-23-01725

Figure Lengend Snippet: Enrichment analysis of the CNKSR2 interactome in the genes harboring neurodevelopmental disease-related de novo variants. CNKSR2 interactors identified in the study were compared with the genes harboring rare de novo variants reported in patients with different neurodevelopmental diseases, and enrichment analysis was performed to determine the association of CNKSR2 interactome with these diseases. ASD: Autism spectrum disorder; CNKSR2: connector enhancer of kinase suppressor of Ras 2; EE: epileptic encephalopathies; ID: intellectual disability; PH: periventricular heterotopia; PMG: polymicrogyria.

Article Snippet: The CNKSR2 antibody (host: rabbit, concentration: 1 mg/mL) used for IP experiments was purchased from Abcepta Biotech (Suzhou, Jiangsu Province, China, Cat# AP69182, RRID: AB_3083424), and rabbit total IgG, used as the negative control antibody, was purchased from Sangon Biotech (Shanghai, China, Cat# D110502, RRID: AB_3083429).

Techniques:

Journal: Aging

Article Title: Pharmacological recapitulation of the lean phenotype induced by the lifespan-extending sulfur amino acid-restricted diet.

doi: 10.18632/aging.206237

Figure Lengend Snippet: Figure 3. The SAAR diet and BSO exert tissue-specific effects on Nrf2 and Phgdh. The SAAR diet increased Nrf2 (A) and Phgdh (B) protein expressions in the liver, which ultimately resulted in higher serine concentrations (C). Unlike the SAAR diet, BSO did not increase Nrf2 and Phgdh in the liver but increased both in the kidneys (D, E). Regardless of the changes in Nrf2 and Phgdh, BSO increased serine concentrations in livers and kidneys (C–F). NAC reversed SAAR-induced changes in Nrf2, Phgdh, and serine (A–F). Note: Sample size = 5-6. Statistical methods are similar to those in Figure 2.

Article Snippet: Host species Antibody dilutions Primary Antibodies Nrf2 Cell Signaling Technology 20733 Rabbit 1:1000 in 5% BSAa Phgdh Cell Signaling Technology 13428 Rabbit 1:1000 in 5% milka β-Actin Sigma A5441 Mouse 1:15000 in 5% milka Vinculin Proteintech 66305-1-Ig Mouse 1:10000 in 5% milka Secondary Antibodies Anti-Rabbit-HRP Cell Signaling Technology 7074 Goat 1:4000 in 5% milk, 1hb Anti-Mouse-HRP Bio-Rad 170-6516 Goat 1:20000 in 5% milk, 30 minb aAll incubations were performed overnight at 4° C. bAll incubations were performed at room temperature. www.aging-us.com 22 AGING Supplementary Table 3.

Techniques: