Journal: Cellular and Molecular Neurobiology

Article Title: Neurotrophic Activity of Cultured Cell Line U87 is Up-Regulated by Proline-Rich Polypeptide Complex and Its Constituent Nonapeptide

doi: 10.1007/s10571-015-0192-8

Figure Lengend Snippet: Western blotting analysis of NGF forms secreted by astrocytoma cell line U87. Supernatants from control and compound-treated cells were separated in SDS-PAGE, then transferred to nitrocellulose membrane. NGF forms were detected with the use of specific polyclonal antibodies anti-βNGF. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed densitometrically in Molecular Imager ChemiDoc MP Imaging System with Image Lab Softwere (BioRad). The representative immunoblot is presented

Article Snippet: Rabbit anti-BDNF polyclonal antibody was from Bioss (MA, USA).

Techniques: Western Blot, SDS Page, Imaging

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: H 2 O 2 inhibited mouse and human TRPM7 current in a [Mg 2+ ] i -dependent manner without affecting expression on the plasma membrane . (A) Time course of whole-cell currents in the presence of various [Mg 2+ ] i in full-length mTRPM7-overexpressing HEK293 cells. Each symbol represents the mean ± SEM of recordings at [Mg 2+ ] i , which are shown on the right. The currents at 2 min (control; black vertical dashed line) and 6 min (H 2 O 2 ; red vertical dashed line) relative to the mean current at 2 min in the absence of Mg 2+ are plotted in . Each symbol represents the mean ± SEM (vertical bar) of 14, 11, 9, 6, 8, 10, 18, 4, and 6 observations in the absence or presence of 2.8 µM, 7.4 µM, 20.9 µM 0.2 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM [Mg 2+ ] i , respectively. (B) Localization of TRPM7 in full-length mTRPM7-wt–overexpressing HEK293 cells. Control conditions (left), 5-min treatment with H 2 O 2 (500 µM; middle), or NMM (100 µM; right). Confocal images show the localization of TRPM7 (green) and nuclei (blue). Scale bar, 20 µm. (C) The effect of oxidative stress induced by H 2 O 2 on the hTRPM7 current. Representative traces of whole-cell currents showing the time course of the current inhibition by H 2 O 2 (500 µM) in the absence (open circles) or presence (closed circles) of 0.2 mM [Mg 2+ ] i in hTRPM7-expressing HEK293 cells. (D) H 2 O 2 (500 µM) inhibited the hTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i ( n = 6), but not in the absence of intracellular Mg 2+ ( n = 5). Each bar represents the mean ± SEM (vertical bar). *, P = 0.00040 versus control. (E) NMM (100 µM) inhibited the hTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i ( n = 5), but not in the absence of intracellular Mg 2+ ( n = 4). Each bar represents the mean ± SEM (vertical bar). *, P = 0.031 versus control.

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Expressing, Clinical Proteomics, Membrane, Control, Inhibition

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Screening of cysteines in mTRPM7 that were responsible for the oxidative stress–induced inhibition. (A) Schematic diagram of 34 cysteines that are conserved in mTRPM7 and hTRPM7. S1107 and D1510 are also depicted (red dots). (B) Expression of TRPM7-wt and its mutants. Immunoblotting of TRPM7 and calnexin (loading control) was performed with HEK293 whole-cell lysates. Numbers on the left represent molecular mass of standards (in kD). (C) The effect of H 2 O 2 (500 µM) on mutant TRPM7 current at 0.2 mM [Mg 2+ ] i . Each bar represents the mean ± SEM (vertical bar). *, P < 0.05 by unpaired t test versus Dox[−] cells. The number of observations ( n ) and exact P values for each mutant are shown in .

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Inhibition, Expressing, Western Blot, Control, Mutagenesis

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Localization of TRPM7 or its mutants in HEK293 cells. Representative confocal images showing the localization of TRPM7 (green) in doxycycline-untreated (Dox[−]) and mTRPM7-wt (WT)–, -C721A–, -C738A–, -C721S–, -C738S–, -C1809S–, and -C1813S–expressing HEK293 cells. Phase-contrast images are also shown. Scale bar, 20 µm.

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Expressing

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Functional expression of the M7cd in HEK293 cells. (A) Time course of M7cd currents in the absence (open circles; n = 15) or presence of 2.8 µM (blue squares; n = 9), 20.9 µM (green triangles; n = 14), and 0.2 mM [Mg 2+ ] i (black circles; n = 16) in HEK293 cells that were treated with doxycycline. Each symbol represents the mean ± SEM (vertical bar). (B) Representative I-V relationship of M7cd current recorded in the presence of various [Mg 2+ ] i at 2 min after break-in. (C) [Mg 2+ ] i -dependent inhibition of M7cd current at 2 min. The current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 µM. Each symbol represents the mean ± SEM (vertical bar) of 15, 36, 19, 27, 14, and 20 recordings in the absence or presence of 0.8 µM, 2.8 µM, 7.4 µM, 20.9 µM, and 0.2 mM [Mg 2+ ] i , respectively. The dashed line is the [Mg 2+ ] i -dependent curve of the full-length TRPM7-wt current (from ). (D) M7cd-S1107E current density was similar in the absence (open inversed triangles; n = 10) and presence of 0.2 mM [Mg 2+ ] i (closed inversed triangles; n = 7). Each symbol represents the mean ± SEM (vertical bar). (E) The effect of H 2 O 2 (500 µM) on M7cd current in the absence of intracellular Mg 2+ (open circles). Each symbol represents the mean ± SEM (vertical bar) of 19 recordings. (F) The time course of the M7cd current in the presence of 0.8 µM [Mg 2+ ] i with (red circles; n = 17) or without (black circles; n = 35) H 2 O 2 application at 2 min. Each symbol represents the mean ± SEM (vertical bar). The current decreased over time even in the control conditions (black circles), and repeated-measures ANOVA revealed that H 2 O 2 has no significant effect on the current decrease (degrees of freedom were corrected using the Greenhouse-Geisser estimate of epsilon; P = 0.247).

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Functional Assay, Expressing, Inhibition, Control

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Functional reconstitution of the TRPM7 current by coexpression of M7kd in M7cd-expressing cells. (A) Transient expression of M7kd-wt in M7cd-expressing cells resulted in an increase in the M7cd current density that was inhibited by H 2 O 2 (500 µM) in the presence of 0.2 mM [Mg 2+ ] i (closed circles; n = 8), but not in the absence of intracellular Mg 2+ (open circles; n = 9). Each symbol represents the mean ± SEM (vertical bar). (B and C) Representative I-V relationships of the M7cd current that was recorded in M7cd-expressing cells transfected with M7kd-wt in the presence (B) or absence of 0.2 mM [Mg 2+ ] i (C), before (black line) or 4 min after (red line) the application of H 2 O 2 (500 µM). (D) [Mg 2+ ] i -dependent inhibition of M7cd with coexpression of M7kd-wt in the control (black squares) and H 2 O 2 -treated (red squares) conditions. The data fit well with a biphasic concentration–response curve, assuming the fraction of high-affinity inhibition of 0.314 estimated for full-length TRPM7-wt (in ). Under control conditions, the current was inhibited by intracellular free Mg 2+ with an IC 50(1) of 7.6 µM and an IC 50(2) of 986 µM. After H 2 O 2 treatment, the current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 µM. Each symbol represents the mean ± SEM (vertical bar) of 28, 9, 13, 16, 19, 12, 5, and 6 observations in the absence or presence of 2.8 µM, 7.4 µM, 20.9 µM, 0.2 mM, 1 mM, 2 mM, and 5 mM [Mg 2+ ] i , respectively. (E) The time course of the M7cd current when M7kd-wt was transfected by either lipofection (closed circles; n = 20) or baculovirus infection (open squares; n = 8) in the presence of 0.2 mM [Mg 2+ ] i . Each symbol represents the mean ± SEM (vertical bar). (F) The effect of H 2 O 2 (500 µM) on the M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of eight recordings. (G) The effect of NMM (100 µM) on M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of six recordings. (H) The effect of coexpression with kinase-inactive mutant M7kd-1645R on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of four recordings. (I) The effect of M6kd coexpression on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of eight recordings.

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Functional Assay, Expressing, Transfection, Inhibition, Control, Concentration Assay, Infection, Mutagenesis

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Schematic illustration of TRPM7 regulation by oxidative stress. Left: Under basal conditions, the current inhibition by Mg 2+ binding to the high-affinity site (indicated by an orange arrow) is attenuated possibly via the interdomain interaction between the channel domain and the kinase domain. The structural integrity of the kinase domain is guaranteed by the zinc-binding motif that coordinates Zn 2+ (indicated by brown) via C1809, C1813, H1750, and H1807. For simplicity, the low-affinity Mg 2+ -binding site that is assumed to exist in the kinase domain is not shown here. Right: Oxidative stress causes the oxidation of the cysteines (C1809 and C1813) in the zinc-binding motif, which may disrupt the interaction between the channel domain and the kinase domain and enhance the inhibition of TRPM7 by intracellular Mg 2+ .

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Inhibition, Binding Assay

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 polymorphisms and the risk of CIN III and cervical carcinoma

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Control

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 and rs2029167 polymorphisms with the risk of HR-HPV positive cervical carcinoma and CIN III

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Control

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 polymorphisms and the risk for CIN III and cervical carcinoma stratified by the sexual, reproductive history

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Infection

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs2029167 polymorphisms and the risk for CIN and cervical carcinoma stratified by the sexual, reproductive history

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Infection

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 and rs2029167 polymorphisms and the risk for cervical carcinoma stratified by clinical pathological characteristics

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques:

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: SMUG1 mRNA expression in CSCCs with different genotypes of rs3087404 and rs2029167 (qPCR).

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: SMUG1 protein expression in CSCCs with different genotypes of rs3087404 and rs2029167 (Western Blot) (A) AA: rs3087404 genotype is AA; AG: rs3087404 genotype is AG; GG: rs3087404 genotype is GG; (B) AA: rs2029167 genotype is AA; AG: rs2029167 genotype is AG; GG: rs2029167 genotype is GG

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a PP5 expression by western blot in fetal (E18), newborn (P1) and adult rat heart tissue. PP5 indexed to GAPDH. Data are mean ± s.e.m., n = 5; * p < 0.05, by two-tailed Student’s t -test. b PP5 expression by western blot in neonatal rat ventricular myocyte (NRVM) cultures under baseline conditions (control) and following treatment with arachidonic acid (aa; 200 µM, 2 h) or okadaic acid (oa, 10 nM, 1 h). PP5 indexed to GAPDH. Data are mean ± s.e.m., n = 5 (three different cell culture batches); * p < 0.05 and ** p < 0.01, by Bonferroni adjusted t -test. c PP5 localization in control, aa-treated, and oa-treated NRVM cultures by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with α-actinin antibody (secondary antibody: FITC-conjugated IgG). The merged image also shows staining of nuclei using Hoechst. Bars, 10 µm. d PP5 localization in control, aa-treated, and oa-treated adult rat cardiomyocyte (ARC) cultures by indirect immunofluorescence. The same antibodies as in c were used. Bars, 5 µm. Right bar graph shows proportion of ARC exhibiting clear PP5 striations or no such striated pattern, for each group. Numbers above columns indicate total number of cells included in the analysis. e Total titin phosphorylation in the three ARC groups measured by ProQ Diamond phosphoprotein vs. Sypro Ruby total protein stain. Bar graph shows mean ± s.e.m., n = 9 (from three independent cell preparations); * p < 0.05, by two-tailed Student’s t -test. f Localization of phosphoserine P-S3991 (titin N2Bus) in control, aa-treated, and oa-treated ARC cultures by indirect immunofluorescence, using anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with α-actinin antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm. Right bar graph shows proportion of ARC exhibiting clear N2Bus P-S3991 striations or no such striated pattern, for each group. Numbers above columns indicate total number of cells included in the analysis

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Expressing, Western Blot, Two Tailed Test, Cell Culture, Immunofluorescence, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence

Journal: BioMed Research International

Article Title: Improvement of Inflammation through Antioxidant Pathway of Gardeniae Fructus 50% EtOH Extract (GE) from Acute Reflux Esophagitis Rats

doi: 10.1155/2020/4826176

Figure Lengend Snippet: Western blot analysis of (a) survivin, (b) cytochrome c, (c) caspase-3, (d) Bax, and (e) Bcl-2 expressions. Nor: normal rats, Con: reflux esophagitis control rats, GL: GE 50 mg/kg treated reflux esophagitis rats, and GH: GE 100 mg/kg treated reflux esophagitis rats. (f) All data are expressed means ± SEM, ( n = 6) rats per group. Significance: ∗ p < 0.05, ∗∗ p < 0.01 versus RE control rat values.

Article Snippet: Mouse monoclonal antibodies against survivin (1 : 1,000, NB 500-205) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Reflux, Control

Journal: Oncotarget

Article Title: Modulation of estrogen related receptor alpha activity by the kinesin KIF17

doi: 10.18632/oncotarget.18104

Figure Lengend Snippet: A. Sequence of the KIF17-Tail. The 12 amino acid NR box peptide containing the LXXLL motif is underlined. B. Co-immunoprecipitation of GFP-ERR1 with myc-EV control, myc-KIF17-T or myc-KIF17T ΔNR expressed in HEK293 cells. Immunoprecipitates and total lysates were analyzed by immunoblot using anti-GFP and anti-myc IgG. C. , D. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in MCF7 cells expressing ERRE-Luc and myc-EV, myc-KIF17-T, myc-KIF17 ΔNR (panel C) or myc-KIF17 NR (panel D). E. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in parental LM2 and LM2 NR cells transfected with ERRE-Luc. F. Same as in panel E, but comparing cells transfected with control or ERR1 siRNAs. Graphs show normalized luminescence values pooled from ≥ 3 experiments performed in triplicate. Error = SEM. *** p < 0.05.

Article Snippet: Lysates were cleared using Protein-G sepharose beads (GE Healthcare) and incubated overnight with 6-8μg rabbit anti-GFP antibody (Novus Biologicals, NB 600-303) or mouse anti-myc antibody (Sigma M4439).

Techniques: Sequencing, Immunoprecipitation, Control, Western Blot, Luciferase, Activity Assay, Expressing, Transfection