Journal: bioRxiv

Article Title: SK2/3 CHANNELS COUPLE WITH T-TYPE CA 2+ CHANNELS TO GATE SPINAL LOCOMOTOR RHYTHM GENERATION

doi: 10.64898/2026.03.19.712770

Figure Lengend Snippet: a-b Representative ventral root recordings showing the effect of the broad-spectrum SK activator 1-EBIO ( a ; 5 mM) and the SK2/3-selective activator CyPPA ( b ; 0.3-0.4 mM) on ongoing fictive locomotion induced by NMDA/5-HT (5/10 µM). Top left in a and b : Schematic of the isolated spinal cord preparation illustrating focal drug puff application onto rhythmogenic segments (L 1 -L 2 ) and bilateral L5 ventral root recordings (L5R, L5L). Bottom: Raw ventral root activity (black) superimposed with low-pass filtered envelopes (blue for 1-EBIO, orange for CyPPA); vertical arrows indicate puff onset. Top right: Expanded time windows ( a’ , b’ ) of the recordings shown below, highlighting rhythm suppression. c-d Representative recordings showing the induction of locomotor-like activity in quiescent preparations (subliminal NMDA/5-HT: 0-1/10 µM) by the SK2/3 inhibitor tamapin ( c ; 1 µM) or the T-type calcium channel blocker nickel ( d ; 10-20 µM). The experimental layout and display are identical to a, b . Low-pass filtered envelopes are shown in purple (tamapin) and teal (nickel). Arrows indicate puff onset.

Article Snippet: Alomone: tamapin (5-10 nM), 1-EBIO (200 μM), CyPPA (1-10 μM), and Tram-34 (5 μM).

Techniques: Isolation, Activity Assay

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Expression and distribution of symplekin in dedifferentiated cells. ( A ) A confluent Caco-2 cell monolayer was scratched with a 1-ml pipette tip. Symplekin (SYM) and a tight junction marker (ZO-1) were immuno-stained at 0 hr and 6 hr after wound healing. Nuclei were stained with DAPI (blue). ( B ) Symplekin and ZO-1 staining in HT-29 cells cultured in glucose-free medium supplemented with galactose (HT-29/gal) or glucose (HT-29/glu). ( C ) Representative WB bands and relative densitometric quantification of total symplekin expression in HT-29/gal and HT-29/glu cells. ( D ) Representative WB bands of cytoplasmic/membrane (Cytosol) and nuclear (Nucleus) distribution of symplekin in HT-29/gal and HT-29/glu cells. Cytoplasmic tubulin and nuclear lamin B1 (LMNB1) were used as controls for quantification analysis, respectively. ( E ) Symplekin expression levels in starved Caco-2 cells treated with EGF for different time periods shown by bands of WB and relative quantitative graphs. ( F ) The localization of symplekin and ZO-1 in Caco-2 cells with (+) or without (−) EGF treatment. Cells were pre-treated with U0126 (40 μM) for 30 min before EGF stimulation. ( G ) Western blot for symplekin in Caco-2 cell fractions with or without EGF or U0126 treatment. The protein levels were normalized to those of tubulin and LMNB1, respectively. * P < 0.05, Student’s t-test. # P < 0.05, one-way ANOVA and Student’s t-test. All data are means ± SD, n = 3. Bars, 20 μm.

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: Expressing, Transferring, Marker, Staining, Cell Culture, Membrane, Western Blot

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Cellular effects induced by the nuclear accumulation of symplekin. ( A , B ) qRT-PCR validation for sucrase and ALP in HT-29 and Caco-2 cells with the indicated treatments. Knockdown of symplekin (ΔSYM) and control (Luc) were also included. MTT assay ( C ) and dual-luciferase assay for Topflash activity ( E ) in HT-29 and Caco-2 cells with the indicated treatments. ( D ) Cell monolayer integrity assessment. The lower chamber medium was collected after 30 min/12 hr permeation for treated HT-29/Caco-2 cells, respectively. Data was analyzed using one-way ANOVA and Student’s t-test. * P < 0.05 versus corresponding differentiation cultured cells. # P < 0.05 versus corresponding RNAi control cells. Values are means ± SEM (n = 3).

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Knockdown, Control, MTT Assay, Luciferase, Activity Assay, Cell Culture

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Transcriptional regulation of cell cycle-related genes by nuclear symplekin coupled with YBX3. ( A ) Representative WB bands and relative densitometric quantification of YBX3 expression in HT-29/gal and HT-29/glu cells. ( B ) Endogenous symplekin was immunoprecipitated to identify its binding capacity to YBX3. IgG was used as a negative control. The protein levels were normalized to those of inputs for quantitative analysis. ( C ) qRT-PCR verification of cell cycle-related genes in HT-29/glu/luc and HT-29/glu/ΔSYM cells. mRNA expression in HT-29/glu/ΔSYM cells was normalized to those in HT-29/glu/luc cells. ( D ) PCR amplification of DNA fragments precipitated by a symplekin antibody and IgG in HT-29/gal and HT-29/glu cells; total sonicated chromatin prior to ChIP was taken as a control. ( E ) q-PCR detection of cyclin D1, cyclin A2, and cyclin B1 promoter enrichment in ChIP DNA products prepared from HT-29/gal and HT-29/glu cells using the indicated antibodies. * P < 0.05 in comparison to the value in HT-29/gal cell with anti-symplekin precipitation, one-way ANOVA and Student’s t-test. # P < 0.05 in comparison to the indicated gene expression in control cells, Student’s t-test. Results are means ± SD, n = 3.

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: Expressing, Immunoprecipitation, Binding Assay, Negative Control, Quantitative RT-PCR, Amplification, Sonication, Control, Comparison, Gene Expression

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Determination of phosphorylated symplekin in cells. ( A ) Cell protein extraction, phosphoprotein purification, and WB assay for symplekin (SYM) in HT-29/gal and HT-29/glu cells. ( B ) WB bands of symplekin in nuclear phosphoproteins isolated from HT-29/gal and HT-29/glu cells. ( C ) Comparison of phosphorylated symplekin between HT-29/gal cytosol and nucleus. ( D ) Comparison of phosphorylated symplekin between EGF stimulated Caco-2 cell cytosol and nucleus. Tubulin and LMNB1 were employed to determine the cell fractionation efficiency. Phosphoproteins were isolated from the eluate fraction. Total cell or nuclear proteins before phospho-purification were used as the input. Phosphorylated symplekin was normalized to the levels of input symplekin in total cellular, cytosolic, or nuclear lysates, and graphic representation of relative amount of phosphorylated symplekin (phospho-SYM) was shown in the bottom panels. # P < 0.05, Student’s t-test. Data are means ± SD, n = 3.

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: Protein Extraction, Purification, Isolation, Comparison, Cell Fractionation

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Phosphorylation and subcellular localization of symplekin regulated by ERK. ( A ) Forward and reciprocal Co-IP between symplekin (SYM) and ERK1/2. WB was carried out with antibodies against symplekin or ERK1/2 in Caco-2 cells with or without EGF treatment. Total cell lysates (input) were used as the loading control for quantitative analysis. ( B , D ) IP using anti-phosphoserine (p-Ser) and phosphothreonine (p-Thr) antibodies in cytoplasmic/membrane (Cytosol) and nuclear (Nucleus) lysates prepared from Caco-2 cells with EGF treatment. The IP input was taken as the normalized control for phosphorylated symplekin quantification. Tubulin and LMNB1 were used as markers for cell fractions. ( C , E ) IP using anti-phosphoserine and phosphothreonine antibodies in nuclear lysates (Nucleus) from Caco-2 cells receiving the indicated treatments. U0126 (40 μM) was added for 30 min before EGF stimulation. The IP input was taken as the loading control for quantitative analysis. LMNB1 was shown as a nuclear marker. * P < 0.05, Student’s t-test. # P < 0.05, one-way ANOVA and Student’s t-test. All data are means ± SD, n = 3. ( F ) IF analyses were performed with an anti-Flag antibody to detect the subcellular localization of symplekin mutants. Nuclei were stained with DAPI. Bars, 20 μm.

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, Control, Membrane, Marker, Staining

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Epithelial junctional complexes affected by symplekin depletion. ( A ) WB bands and quantification analysis for expressions of occludin, E-cadherin, β-catenin, and DSG2 in stable symplekin-silenced HT-29 cells (Sh1 and Sh2) and the control (Luc). ( B , C ) IF staining of E-cadherin and β-catenin in the above stable clones. DAPI showed the nuclei staining. ( D ) Cytoskeletal F-actin staining using rhodamine phalloidin. ( E ) Morphological comparison among indicated HT-29 cell colonies captured by a phase-contrast microscopy. ( F ) Matrigel invasion assays in HT-29 cells with or without symplekin knockdown. The migrated cells were stained with crystal violet and counted in six random fields. FV, field vision. ( G ) Cell disassociation and aggregation analyses. Six random fields were chosen for each group. * P < 0.05, one-way ANOVA and Student’s t-test. All results are means ± SD, n = 3. Bars, 20 μm.

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: Control, Staining, Clone Assay, Comparison, Microscopy, Knockdown

Journal: Scientific Reports

Article Title: Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner

doi: 10.1038/s41598-017-04005-z

Figure Lengend Snippet: Schematic representation of the ERK-dependent regulation of symplekin function.

Article Snippet: Antibodies were purchased from the following companies: two symplekin monoclonal antibodies (mAbs) were from Progen Biotechnik (cat #651100; Heidelberg, Germany) and BD Biosciences (cat #610644; Rockville, MD, USA); ZO-1 mAb (HPA001637), CSDA mAb (SAB1404593), GAPDH mAb (G8795), rabbit anti-FLAG (F7425) antibody, FITC-conjugated goat anti-rabbit Immunoglobulin G (IgG) antibody (F6005), anti-flag affinity gel (F2426), and 3 × FLAG Peptide (F4799) were from Sigma-Aldrich (St. Louis, MO, USA); ERK1/2 mAb (13–6200) and rabbit anti-β-catenin (AHO0462) antibody were from Invitrogen (Carlsbad, CA, USA); biotinylated rabbit anti-phosphoserine (ab9335) and rabbit anti-phosphothreonine (ab9340) antibodies were from Abcam (Cambridge, MA, USA); rabbit anti-E-cadherin (3195S) antibody was from Cell Signaling Technology (Beverly, MA, USA); tubulin-α mAb (66031) and LMNB1 mAb (66095) were from Proteintech (Wuhan, China); Cy3-conjugated goat anti-mouse IgG antibody (115-165-166) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); peroxidase-conjugated goat anti-mouse IgG antibody (HD003-1) was from DingGuo (Beijing, China); and peroxidase-conjugated goat anti-rabbit IgG antibody (M21002) was from Abmart (Shanghai, China).

Techniques: