Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 stimulates MEF2D/PGC1α/Nrf2 signal pathway through regulation of the Akt/GSK3β pathway in PD animal models. ( A ) Representative Western blots and densitometric analysis the expression of CDK5, p-MEF2D, MEF2D and PGC1α. ( B , E ) Representative Western blots and densitometric analysis of the expression of p-Akt and p-GSK3β. ( C , F ) Representative Western blots and densitometric analysis of the expression of Nrf2, HO-1 and TFAM. ( D ) Representative Western blots and densitometric analysis the expression of MEF2D and PGC1α. Data are expressed as mean±SEM (n=3 per group). # P<0.05, ## P<0.01, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 and *** P<0.001vs. MPTP or 6-OHDA group.

Article Snippet: CGNs of 1.0-1.5×10 5 were transfected with MEF2D siRNA (Santa Cruz, CA) and scramble siRNA (Santa Cruz, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as followed to the manufacturer’s instructions.

Techniques: Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 prevents the loss of SN DA neuron loss by activating MEF2D/PGC1α/GSK3β signal pathway. Representative images of middle brain sections co-stained with antibodies against ( A , D ) TH (green) and p-GSK3β (red); ( B , E ) TH (red) and MEF2D (green); ( C , F ) TH (green) and PGC1α (red). DAPI (blue) indicates nucleus. ( G , H ) Quantitative analysis of immunofluorescence intensity in TH-positive cells. Data are expressed as mean±SEM (n=3 to 4 per group). # P<0.05, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 vs. MPP + or 6-OHDA group.

Article Snippet: CGNs of 1.0-1.5×10 5 were transfected with MEF2D siRNA (Santa Cruz, CA) and scramble siRNA (Santa Cruz, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as followed to the manufacturer’s instructions.

Techniques: Staining, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 activates the MEF2/PGC1α/Nrf2 pathway through regulation of the Akt-GSK3β pathway. A53T and corrected DA neurons treated with T-006 and the positive drug at the indicated concentration for 24 hours. For MPP + -treatment assay, CGNs were pretreated with or without LY294002 (1 μM), Akt-iv (1 μM), or LiCl (10 μM) for 2 h, incubated with or without T-006 for 2 h, and finally exposed to MPP + . Cell viability was examined using an MTT assay. Luciferase reporter gene assays respectively included MEF2 ( A ), PGC1α ( B ), PGC1α-ΔMEF2 ( C ) and ARE ( D ). ( E ) Representative images of neurons co-stained with antibody against Nrf2 (green). DAPI (blue) indicates nucleus. ( F ) Luciferase reporter gene assays of MEF2 with MPP + induction. ( G – K ) respectively represent the fold changes of CDK5, MEF2D, PGC1α, Nrf1 and Nrf2 at mRNA level. ( L ) Effect of MEF2D reduction on MPP + -induced neurotoxicity in CGNs. ( M ) Effects of Akt pathway inhibitor LY294002 and GSK3β inhibitor on MEF2 transcriptional activity. ( N ) Effects of Akt pathway inhibitors LY294002 and Akt-iv, and GSK3β inhibitor on MPP + -induced neurotoxicity in CGNs. Data above are all from three independent experiments, expressed as mean±SEM. # P<0.05, ## P<0.01 and ### P<0.001 vs. control (Ctrl) group, and * P<0.05, ** P<0.01, *** P<0.001 vs. MPP + group.

Article Snippet: CGNs of 1.0-1.5×10 5 were transfected with MEF2D siRNA (Santa Cruz, CA) and scramble siRNA (Santa Cruz, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as followed to the manufacturer’s instructions.

Techniques: Concentration Assay, Incubation, MTT Assay, Luciferase, Staining, Activity Assay, Control

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: a Mef2D is extensively disordered and predicted to form liquid-liquid phase separated condensates. The Mef2D structure predicted by Alphafold indicates a small structured domain involving the N-terminal ~100 residues and most of the transactivation domain (TAD) contains is disordered. The regions promoting formation of liquid-like droplets by the FuzDrop method are marked by blue. The β-domain (magenta) appears as an ordered motif within the disordered transactivation region. FuzDrop predictions shown on the right panels indicate high droplet-promoting probability (p DP ) in particular for regions 155-268 residues and 341-520 residues, which are predicted to spontaneously form liquid-liquid phase separated condensates. The β-domain (magenta) and its flanking regions (cyan) are predicted to serve as ordered interaction motifs within the condensate (see also Supplementary Fig. ). In addition, the β-domain region is capable of sampling a multiplicity of binding modes (MBM), indicating its sensitivity to the cellular context. b Sequences of the designed Mef2D variants. The β-domain and its flanking regions are shown for the wild-type ( wt ) Mef2D (UniProt code: Q14814; https://legacy.uniprot.org/uniprot/Q14814 ; 265-301 residues), var1 and var2 with similar β-domain dynamics (gray), var3 and var4 with mobile β-domains (green), var5 - var8 with rigid β-domains (red) as compared to wild-type Mef2D. The sequence of the β-domain is magenta, mutated residues (orange) are highlighted. c Predicted β-domain disorder of Mef2D variants. Structural disorder in the unbound state of Mef2D were computed for the full protein sequence using the Espritz method as embedded in the FuzPred program and the p D values were averaged for residues 286-292. The var3 and var4 variants (green) are above the threshold between disorder and order (p D ≥ 0.3085 ). var1 (gray) has similar, var2 (gray) has slightly more mobile β-domain than the wild-type Mef2D (black). var5 - var8 variants (red) are predicted to have more rigid β-domain than the wild-type. d Droplet landscape of the Mef2D variants. The droplet landscape shows the droplet probability (p DP ) as a function of the multiplicity of binding modes (MBM) , . The assemblies below the diagonal are likely more solid-like, those above the diagonal are more liquid like . High MBM values indicate an increased likelihood to change between liquid-like and solid-like forms, for example in case of var8 . More mobile β-domain variants ( var3 , var4 , green diamonds) exhibit increased probability to form droplets (higher p DP ), whereas more rigid β-domain variants ( var5 - var8 , red triangles) more likely form solid-like states depending on the cellular conditions (high MBM).

Article Snippet: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Sampling, Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: Luciferase activity normalised to galactosidase signal (Methods) is shown as a percentage of the wild-type (wt) control. Luciferase activity was measured in four biologically independent experiments, using three technical replicates in each with the same samples ( n = 12 samples in 4 independent experiments). The points represent individual measured data, the rectangles in the box plots present the median and the 25 and 75 percentile values, while the error bars point to 1 and 99%. The luciferase signal is shown as mean ± SE, significance (* p < 0.05; ** p < 0.01; # p < 0.005; ## p < 0.001) was computed using two-sided student t-test. The different variants are grouped by their β-domain dynamics properties (Fig. , Methods): var1 (gray diamond) and var2 (gray triangle) with similar β-domain dynamics; var3 (green diamond) and var4 (green triangle) with mobile β-domain; var5 (red diamond), var6 (red triangle), var7 (red circle) and var8 (red square) with rigid β-domain as compared to the wild-type. The β- variant is shown by blue diamond. a Transcriptional activity in non-differentiated C2C12 myoblasts. Variants with rigid β-domains show significantly higher transcriptional activity then the wild-type ( var5 178 ± 9.6, var6 135.6 ± 9.3, var7 141.6±8.1 and var8 140.8±11.5 %), while variants with mobile β-domains show slightly increased transcription activity ( var3 127.7 ± 4.9 and var4 123.9 ± 5.5 %) using n = 12 samples in 4 independent experiments. Significances ( var3 p = 0.0001, var4 p = 0.0012, var5 p = 5.2*10 −6 , var6 p = 0.0027, var7 p = 0.0003, var8 p = 0.0044; β-minus p = 1.52*10 −6 ) were computed using two-sided student t-test. b Transcriptional activity in differentiated C2C12 myotubes . var8 with rigid β-domain (red square) exhibits significantly higher transcriptional activity than the wild-type, while var3 (green diamond) and var4 (green triangle) with mobile β-domains, and var2 (gray triangle) with similar β-domain dynamics as the wild-type exhibit reduced transcriptional activity. ( n = 12 samples in 4 independent experiments). Significances ( var2 p = 2.9*10 −9 ; var3 p = 0.008; var4 p = 1.02*10 −7 ; var8 p = 0.0004) were computed using two-sided student t-test. c Lack of Mef2D blocks myotube formation in Mef2D knockout C2C12 cell line. Western blot images showing the lack of Mef2D, which are present endogenously in C2C12 cells. Three chosen stable KO cultures were followed through several passages to prove the stable lack of Mef2D (~70 kDa), while actin was used as inner control (~40 kDa). After 6 days of differentiation myotubes form in C2C12 cell line with endogeneous Mef2D (control, left panel), while cannot be observed in MEF2D KO cultures (right panel). Images were taken by transmitted microscopy, scale bars represent 400 µm. d Transcriptional activity in C2C12 KO cells. Rigid β-domain variants (red) have higher transcriptional activity than variants with similar dynamics to the wild-type (gray). Significances ( var4 p = 0.0175, var5 p = 0.0096, var6 p = 0.0365; var7 p = 0.0022; var8 p = 0.0071) were computed using two-sided student t-test using n = 9 samples in 3 independent experiments).

Article Snippet: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Luciferase, Activity Assay, Control, Variant Assay, Knock-Out, Western Blot, Microscopy

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: a Early stage of myotube development (day 2 - day 4). The number of multinucleated, long myotubes in the presence of overexpressed Mef2D variants. Representative fluorescent and transmitted images represent randomly selected visual fields and were used to determine the fusion index of the appropriate cultures. Each experiment was independently repeated two times with similar results, at least 15 randomly selected visual fields were analysed. Scale bar is 50 µm. b , c Protein expression of myogenic regulatory factors MyoD ( b ) and Desmin ( c ). Normalized protein expression during the differentiation of C2C12 cells. Protein expression was plotted as a percentage of their wild-type control in each day of differentiation. Data was derived from quadruplicate measurements ( n = 4 independent experiments), significances (* p = 0.0032; ** p = 0.0172 for panel b , and * p = 0.0149; ** p = 0.0002 for panel c ) were computed using two-sided student t-test as compared to the wild-type control on the given day of differentiation. b Protein expression of the early differentiation regulator MyoD. Variants with rigid β-domains ( var5 - var8 , red) exhibit higher level of MyoD expression on day 1 and day 2. Significant deviations ( var5 p = 0.0032; var8 p = 0.0172) were observed in case of var5 and var8 using two-sided student t-test as compared to the wild-type control on the given day of differentiation. c Protein expression of the late differentiation marker desmin. More rigid β-domain variants ( var5 p = 0.0149; var8 p = 0.0002, red) significantly increase desmin expression on days 1 and 2.

Article Snippet: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Expressing, Control, Derivative Assay, Marker

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: a Mef2D foci in the nucleus and cytoplasm. Subcellular distribution of Mef2D wt , var3 , var4 and var8 in C2C12 cells grown in cycling or differentiating medium exhibit foci formation in both the nucleus and cytoplasm. Higher-order assembly is most pronounced in case of var8 with rigid β-domain, but is also observed in case of var3 and var4 with mobile β-domain. 24 hrs post-transfection cells were fixed and stained with an antibody specific for Mef2D. Nucleic acid was stained using DAPI. The scale bar is 10 μm on the representative images. The experiment was performed four times (cycling medium) and three times (differentiating medium). Quantification is shown in panel b . b Quantification of MEF2D cells with cytoplasmic higher-order structures (foci). The percentage of Mef2D overexpressing cells with cytoplasmic aggregates is significantly higher in case of var8 with rigid β-domain. Cycling C2C12 cells: n = 4, ± s.e.m.; differentiating C2C12 cells: n = 3 independent experiments, ± s.e.m. Total number of cells counted > 150. Significances were computed by one-way ANOVA followed by Bonferroni-Holm Posthoc in reference to wt : var3 p = 0.13 (cycling) and p = 0.87 (differentiated), var4 p = 0.09 (cycling) and p = 0.60 (differentiated), var8 p = 0.04 (cycling) and p = 0.04 (differentiated). c , d Analysis of mobility of Mef2D higher-order assemblies in nuclear foci ( c ) and cytoplasmic foci ( d ). Mobility was assessed by fluorescence recovery after photobleaching (FRAP) performed after 24 hours post-transfection of GFP-tagged MEF2D wt, var3 , var4 and var8 in C2C12 cells. The mean of the FRAP curve +/- standard error of the mean (s.e.m.) is shown. c Number of nuclear foci analyzed: wt (3); var3 (3); var4 (3); var8 (3). d number of cytoplasmic aggregates analyzed: wt (11); var3 (10); var4 (10); var8 (9). All Mef2D proteins show high mobility inside the nuclear foci ( c ) and nucleoplasm (Supplementary Fig. ). This is in sharp contrast with the low mobility inside cytoplasmic foci ( d ).

Article Snippet: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Transfection, Staining, Fluorescence

Journal: Nature Communications

Article Title: Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

doi: 10.1038/s41467-023-37017-7

Figure Lengend Snippet: Conformational analysis was performed using the 70-100 ns trajectory of each replica (9000 snapshots) (Methods). a Contacts maps of the clusters. Mef2D wt , var3 , var4 and var8 peptides exhibit distinct intra-molecular interaction patterns (Methods). The β-domain (blue) contributes to structure organisation of var8 and to lesser extent to var4 , while does not form persisting contacts in var3 (see also Supplementary Fig. ). Color scales indicate the number of snapshots in the clusters, with the given contact sampled. b Representative structures of the of Mef2D variant peptides. More compact structures are formed through interactions of the β-domain (blue), such as in case of var8 and var4 , while extended structures, such as in case of var3 sample variable interactions outside the β-domain. β-domain residues are displayed in blue, residues mutated in the different variants are orange labelled.

Article Snippet: C2C12 cells were transfected with MEF2D specific CRISPR/Cas9 knockout and HDR plasmid constructs, targeting 3 different places in coding sequence (in exon# 3, 4 and 5; Santa Cruz Technology, Dallas, TX, USA).

Techniques: Variant Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 5. Dex reversed H/R-induced upre gulation of miR-665 and downregulation of MEF2D. (A-B) Expressions of miR-665 and MEF2D mRNA were examined by qRT-PCR. (C-D) Expressions of MEF2D protein were detected by Western blot. Quantitative analyses of protein band intensity. GAPDH served as an internal control for sample loading. (E) Predicted duplex formation between MEF2D 3′- UTR and miR-665. (F) Dual luciferase gene reporter assay manifested that miR- 665 could directly bind with MEF2D. (n = 3 per group). **p < 0.01, ***p < 0.001, ****p < 0.0001. qRT- PCR, quantitative reverse transcription PCR; UTR, untranslated region; MEF2D, myocyte enhancer factor 2D.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Quantitative RT-PCR, Western Blot, Control, Luciferase, Reporter Assay, Reverse Transcription

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 6. Dex improved cell viability and decreased apoptosis of H9c2 cells undergoing H/R via downregulation of miR-665 followed by upregulation of MEF2D. The H9c2 cells were transfected with miR-665mimics or co-transfection of miR-665mimics with pcDNA-MEF2D for 48 h before treatment with 10 nM Dex for 1 h. (A) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on morphology of H9c2 cells in each group were observed using an inverted microscope (scale bars, 100 µm). (B) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on the cell viability were detected using CCK-8 assay. (C-F) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on the cell apoptosis were gauged by flow cytometry. The apoptotic rates were presented as addition of the percentages of cells at early apoptotic phase and late apoptotic phase. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Transfection, Cotransfection, Over Expression, Inverted Microscopy, CCK-8 Assay, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 7. Dex protected against pyroptosis of H9c2 cells undergoing H/R via downregulation of miR-665 followed by upregulation of MEF2D. (A) Protein bands of MEF2D, IL-1β, IL-18, NLRP3, ASC, C-Caspase-1, and GSDMD were evaluated by Western blot. (B-C) Expressions of miR-665 and MEF2D mRNA were detected by qRT- PCR. (D-J) Quantitative analyses of protein band intensities of MEF2D, IL-1β, IL-18, NLRP3, ASC, C-Caspase-1, and GSDMD. GAPDH served as an internal control for sample loading. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 8. Dex facilitated nuclear translocation of Nrf2 regulated by MEF2D in H/R-treated H9c2 cells. (A) Expressions of cytoplasmic Nrf2 and nuclear Nrf2 proteins were determined by Western blot in H/R-treated H9c2 cells subjected to Dex pretreatment. GAPDH and Lamin B were used as internal references for sample loading respectively. (B-C) Quantitative analyses of the expression levels of cytoplasmic Nrf2 and nuclear Nrf2. (D) Expressions of cytoplasmic Nrf2 and nuclear Nrf2 proteins were detected by Western blot in H/R-treated H9c2 cells undergoing Dex pretreatment and transfection of miR-665mimics or co-transfection of miR-665mimics with pcDNA-MEF2D respectively. GAPDH and Histone3 acted as internal references for sample loading respectively. (E-F) Quantitative analyses of the expression levels of cytoplasmic Nrf2 and nuclear Nrf2. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant. Nrf2, nuclear factor erythroid 2-related factor 2.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Translocation Assay, Western Blot, Expressing, Transfection, Cotransfection

Journal:

Article Title: The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation

doi:

Figure Lengend Snippet: Exogenous stable expression of GRIP-1 sense and antisense in C2C12 myogenic cells demonstrates that GRIP-1 expression is necessary for terminal skeletal muscle cell differentiation. Total RNA was isolated from both normal C2C12 and stably transfected C2:GRIP-1 sense and antisense cells at the PMB and CMB stage (∼50% and 100% confluent, respectively) cultured in growth medium, and at a stage 72 hr/3 days after the withdrawal of serum (i.e., propagated in differentiation medium, MT-3). RNA samples were blotted and probed with 32P-radiolabeled cDNA encoding GAPDH, myoD, myogenin, cyclinD1, and p21. MEF-2 proteins were detected by Western blot with 30 μg of nuclear proteins from each sample using a rabbit antibody to MEF-2 (Santa Cruz C-21), which was generated against MEF-2A but which cross-reacts with mouse/human MEF-2A, MEF-2C, and MEF-2D. (GM) Growth medium (DMEM containing 20% FCS); (DM) differentiation medium (DMEM containing 2% horse serum).

Article Snippet: MEF-2 proteins were detected by Western blot with 30 μg of nuclear proteins from each sample using a rabbit antibody to MEF-2 (Santa Cruz C-21), which was generated against MEF-2A but which cross-reacts with mouse/human MEF-2A, MEF-2C, and MEF-2D. (GM) Growth medium (DMEM containing 20% FCS); (DM) differentiation medium (DMEM containing 2% horse serum).

Techniques: Expressing, Cell Differentiation, Isolation, Stable Transfection, Transfection, Cell Culture, Western Blot, Generated