Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 1 Glomerular TMEM14A expression was diminished before onset of proteinuria. (a) Urinary albumin excretion is significantly higher in Dahl rats compared to SHR from 6 weeks of age. Y-axis shows urinary albumin excretion in mg/24 h, and the X-axis shows animal age in weeks. (b) Glomerular TMEM14A mRNA expression in Dahl rats is significantly lower than in SHR controls at all time points. The Y-axis shows mRNA expression as relative expression compared to Hprt1, and the X-axis shows animal age in weeks. No correlation was observed between TMEM14A expression and urinary albumin excretion in both SHR (r = − 0.80, CI −0.98 to 0.27) and Dahl (r = −0.60, CI −0.97 to 0.60) using Pearson's correlation coefficient. (c) Glomerular staining for Tmem14a protein is lower in Dahl rats than in SHR after 2 weeks. This difference is significant at Weeks 4 and 8 of age. The columns represent mean semiquantitative score. Dahl rats develop proteinuria at 6 weeks of age. Thus, both mRNA and protein expression levels drop before onset of proteinuria. ANOVA with Tukey's post hoc analysis was used.(d–m) Representative images of glomeruli of spontaneously proteinuric Dahl (d–h) and SHR (i–m) rats at respectively 2 (d and i), 4 (e and j), 6 (f and k), 8 (g and l), and 10 (h and m) weeks of age. (a–c) Horizontal lines indicate mean with SD. Scale bar = 50 μm *p < 0.001.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: Expressing, Staining

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 2 Knocking down TMEM14A mRNA translation causes proteinuria. Knocking down mRNA translation of the zebrafish homologue of TMEM14A through morpholino injection results in proteinuria. (a–f) Representative immunofluorescence images of transversal sections of zebrafish proximal tubule cells after injection of a mixture of red labeled 3 kDa dextran tracer (a, c, e) and green labeled 70 kDa dextran tracer (b, d, f) in controls (a,b), TMEM14A knockdowns (C and d), and PAN injected positive controls (e,f). Dextran tracers that passed the GFB are reabsorbed by proximal tubule epithelial cells in endosomes. Thus, reabsorbed dextran tracer appears as fluorescent droplets. The number of proximal tubule reabsorption droplets was counted in a blinded manner in sections as those shown here. The arrowheads in B point out examples of counted droplets. The circled areas show high fluorescence due to dextran present in the peritubular capillaries. These areas are not counted as reabsorption droplets. The sharpness of the images in panels A through F has been enhanced by overlaying them with a digital high pass filter. (g and h) Uptake of the red 3 kDa marker (g) was used to assess tubular reabsorption function, which was intact in both TMEM14A knockdown animals and controls. In the knockdown model, significantly more 70 kDa droplets (h) have passed the GFB and were subsequently reabsorbed. Puromycin aminonucleoside (PAN) injected zebrafish were used as positive controls. Students t-test was used.(g and h) Horizontal lines indicate mean with SD. Scale bar = 20 μm.* = p < 0.05.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: Injection, Immunofluorescence, Labeling, Fluorescence, Marker, Knockdown

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 3 TMEM14A was primarily expressed by podocytes. In vitro experiments of relative TMEM14A mRNA expression show expression relative to GAPDH expression when comparing mRNA extracts from whole kidney, purified glomeruli, human umbilical vein endothelial cells (Huvec), human embryonic kidney (HEK), and finally podocytes. Expression in podocytes, HEK and Huvec was significantly higher than in whole kidney (p < 0.001 for all groups) and then purified glomeruli (p < 0.001 for podocytes and HEK, p < 0.05 for Huvec). Podocyte expression was highest of all cells and significantly so compared to Huvec (p < 0.05). Mean and SD shown. ANOVA with Tukey's post hoc analysis was used.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: In Vitro, Expressing, Purification

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 4 Glomerular TMEM14A expression was increased in human proteinuric renal diseases. (a) Glomerular TMEM14A protein expression was examined in human kidney biopsies from patients with diabetic nephropathy (DN), lupus nephritis (LN), IgA nephropathy (IgAN), minimal change disease (MCD), and healthy controls. Compared to controls, TMEM14A protein expression is significantly more extensive in IgAN, LN, and MCD, but not in DN. (b–f) Representative images of glomeruli stained for TMEM14A in healthy controls (b), diabetic nephropathy (c), lupus nephritis (d), IgA nephropathy (e), and minimal change disease (f). Slides were stained with goat anti-TMEM14A antibody and immunoreactivity was assessed by diaminobenzidine. This results in a brown color which then indicates TMEM14A localization. Counterstaining with hematoxylin results in blue-purple coloring of cell nuclei. Boxes in a show the range of values between the lower and upper quartile, the whiskers show 5th–95th percentile, triangles show values lying outside the 5th–95th percentile, the line in the box shows the median, and “+” indicates the mean. The scale bar in B applies to B through f and indicates 50 μm. * = p < 0.001. ANOVA with Tukey's post hoc analysis was used.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: Expressing, Staining

Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: Inhibition of Cdc25A/Cdk–cyclin interactions by Thr504 phosphorylation. (A) GST-Cdc25A fusion proteins (wild type (WT), T504A, or C428S) were first incubated with GST-Δ60-Chk1 (Δ60) or its kinase-dead form (DA) and then with Tyr15-phosphorylated Cdk1–cyclin A1 complexes. The reaction mixture was then analyzed by immunoblotting using anti-Cdc25A, anti-phospho-Thr504, anti-cyclin A1, or anti-Cdk1 phospho-Tyr15 antibodies. (For exact experimental conditions, see Supplementary data.) (B) Activated eggs were injected with 2 ng of mRNA encoding GST (Cont.) or GST-Cdc25A (WT or T504A), incubated for 2.5 h, reinjected or not with 2 ng of Δ60-Chk1 mRNA, and then incubated further for 1.5 h. Egg extracts (Input; equivalent to half an egg) and GST-pulled down proteins (GST-PD; equivalent to 10 eggs) were then immunoblotted for GST-Cdc25A or endogenous cyclins A1, B1, or E1 (left panel). (The blot of GST-Cdc25A in GST-PD is short-exposed.) The levels of cyclins (in both Input and GST-PD) were quantified using the NIH Image program from four independent experiments, and the levels of cyclins bound to GST-Cdc25A proteins were normalized to the input of cyclins; values obtained for WT Cdc25A (−Chk1) were set at 1.0 (right panel). (C) Extracts from the eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in (B) were mixed with extracts from the eggs expressing Myc-tagged cyclins A1 or B1 (together with Xe-Wee1B (Okamoto et al, 2002) to induce Cdk1 Tyr15 phosphorylation), incubated for 1 h at 4°C, and analyzed for GST-Cdc25A or ectopic cyclins A1 or B1 as in (B, left). (D, E) Activated eggs were injected with 2 ng of mRNA encoding wild-type GST-Cdc25A, GST-Cdc25A-ΔC23 (D) or GST-Cdc25A-3A (E), incubated for 2.5 h, and analyzed as in (B). In (B–E), all the GST-Cdc25A constructs had both S73A and C428S mutations (see text).

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Inhibition, Incubation, Western Blot, Injection, Expressing, Construct

Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: Requirement of Cdc25A phosphorylation on Thr504 for the DNA replication checkpoint. (A, B) Activated eggs were injected with 1 ng of mRNA encoding Myc-tagged wild-type Cdc25A or indicated Myc-tagged Cdc25A mutants, reinjected 2.5 h later with 2 ng of Δ60-Chk1 mRNA, and then analyzed by immunoblotting using anti-Myc or anti-Cdk1 phospho-Tyr15 antibodies. (C–E) One-cell embryos were uninjected (Cont.) or injected with 1 ng of mRNA encoding either wild-type Cdc25A or T504A Cdc25A, cultured, and analyzed for immunoblotting (after stage 8 with anti-Cdc25A or anti-Cdk1 phospho-Tyr15 antibodies; C), for the external morphology (D) and for the percentage embryonic death at stage 11 (E).

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Injection, Western Blot, Cell Culture

Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: No requirement of 14-3-3 binding for the inhibitory effect of Thr504 phosphorylation. (A) Activated eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in Figure 4B were subjected to GST pulldown assays and analyzed by immunoblotting using anti-GST antibody or anti-human 14-3-3β antibody (which can recognize all 14-3-3 isoforms). (B) Chk1-phosphorylated GST-Cdc25A protein (wild type) was GST-pulled down from activated eggs, washed with or without 0.4% Empigen, and analyzed by immunoblotting as in (A) (left). Extracts from the eggs expressing GST-cyclin A1 (together with Xe-Wee1B; see Figure 4C legend) were treated with the indicated peptide-coupled beads and immunoblotted for cyclin A1 or 14-3-3 protein (right). See Materials and methods for details. (C) GST-Cdc25A proteins (phosphorylated or not by Chk1) and egg extracts (equivalent to 10 eggs) were both prepared as in (B) (GST-Cdc25A proteins being eluted from glutathione beads), mixed together, and incubated for 1 h at 4°C. GST-Cdc25A proteins were then immunoprecipitated with anti-Cdc25A antibody (IP) and analyzed by immunoblotting using anti-GST or anti-cyclin A1 antibodies. As control of GST-Cdc25A proteins (wild type or T504A), GST alone was used (Cont.). (D) GST-Cdc25A proteins prepared as in (C) were first incubated or not with 0.1 μg of recombinant Xenopus 14-3-3ɛ protein in 30 μl of an egg extraction buffer for 15 min at 4°C and then with (GST-)Cdk1–cyclin A1 complexes (purified from 10 eggs) for 1 h. (Xenopus 14-3-3ɛ protein can bind to phosphorylated Thr504 of Cdc25A; see Materials and methods.) The mixture was then analyzed as in (C). In (A–D), all the GST-Cdc25A constructs had both S73A and C428S mutations.

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Binding Assay, Expressing, Western Blot, Incubation, Immunoprecipitation, Recombinant, Purification, Construct

Journal: AIDS Research and Human Retroviruses

Article Title: Proteasomal Inhibition Potentiates Latent HIV Reactivation

doi: 10.1089/aid.2020.0040

Figure Lengend Snippet: FIG. 4. Bortezomib increases Cyclin T1 and activates NF-jB. (A) Human PBMCs were stimulated for 24 h with DMSO and bortezomib. Nuclear extracts were prepared and run on 10% SDS-PAGE. Membranes were probed for anti-human CycT1 and b-actin. Densitometry was performed and normalized to b-actin, and the expression in untreated controls was set to 1. All samples represented were run on the same gel (space indicates lanes omitted from the figure). (B) Human PBMCs were stimulated for 9 h with DMSO, PMA and PHA, MG132, and bortezomib at indicated concentrations. Nuclear extracts were prepared and run on 10% SDS-PAGE. Membranes were probed for anti-human ph-IjBa, total IjBa, and b-actin. Densitometry was performed and normalized to total IjBa and b-actin, and the expression in untreated controls was set to 1. (C) Human PBMCs were stimulated for 1 h with DMSO, PMA and PHA, MG132, TNFa, and bortezomib at indicated concentrations. Nuclear extracts were prepared and run on 10% SDS-PAGE. Membranes were probed for ph-p65, total p65, and b-actin. Densitometry was performed and normalized to total p65 and b-actin, and the expression in untreated controls was set to 1. Results are representative of Western blots from three healthy donors. NF-jB, nuclear factor jB; PBMCs, peripheral blood mononuclear cells; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TNFa, tumor necrosis factor alpha.

Article Snippet: Membranes were blocked in 5% nonfat milk (NFM) for at least 1 h and blotted overnight with mouse anti-human CycT1 antibody (sc-271348; Santa Cruz Biotechnology), rabbit anti-human p-p65 (3033S; Cell Signaling), rabbit anti-human p65 (8242S; Cell Signaling), mouse antihuman p-IjBa (9246S; Cell Signaling), mouse anti-human IjBa (48145S; Cell Signaling), and rabbit anti-human b-actin (ab8227; Abcam) antibodies in 5% NFM.

Techniques: SDS Page, Expressing, Western Blot, Polyacrylamide Gel Electrophoresis