Journal: The Journal of Cell Biology

Article Title: Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

doi: 10.1083/jcb.200107037

Figure Lengend Snippet: Immunoblot analysis of smitin relatedness to titin and c-titin and smitin expression in smooth muscles. (A) Reactivities of titin and smitin with a polyclonal antibody raised against gizzard smitin and a monoclonal antibody raised against mammalian striated muscle titin. SDS-extracts of chicken pectoralis striated muscle (lanes 1, 3, and 5) and chicken gizzard smooth muscle (lanes 2, 4, and 6) were subjected to SDS-PAGE. One pair of lanes was stained with Coomassie blue (lanes 1 and 2). Two other sets of lanes were electroblotted to nitrocellulose (lanes 3–6). One blotted set of lanes was incubated with a rabbit polyclonal antibody raised against injected chicken gizzard smitin and developed (lanes 3 and 4). The other blotted set of lanes was incubated with the 9D10 mouse monoclonal antibody reactive against vertebrate striated muscle titin and developed (lanes 5 and 6). The blots were developed using Western analysis with an alkaline phosphatase–conjugated secondary antibody. (B) Reactivities of smitin and chicken intestinal epithelial cell c-titin with the anti-smitin polyclonal antibody. SDS extracts of chicken aorta smooth muscle (lanes 1 and 3) and chicken intestinal epithelial cell brush borders (lanes 2 and 4) were subjected to SDS-PAGE. Lanes from the gel were stained with Coomassie blue (lanes 1 and 2) or electroblotted to nitrocellulose, incubated with the anti-smitin polyclonal antibody, and developed for Western analysis (lanes 3 and 4). (C) SDS extracts of chicken pectoralis striated muscle (lanes 1 and 5), gizzard smooth muscle (lanes 2 and 6), intestinal smooth muscle (lanes 3 and 7), and aorta smooth muscle (lanes 4 and 8) were subjected to SDS-PAGE. The gel was stained with Coomassie blue (lanes 1–4) or blotted to nitrocellulose (Lanes 5–8). The blot was probed with an anti-smitin polyclonal antibody by Western analysis. A, actin; M, myosin heavy chain; Smt, smitin; T, titin.

Article Snippet: For double-label immunofluorescence analysis, the gizzard smooth muscle sections were incubated simultaneously in the anti-smitin polyclonal antibody and an anti-pan myosin monoclonal antibody (BAbCO MMS-456S; Innovative Products), and the cardiac muscle was incubated with the anti-smitin polyclonal antibody and the 9D10 anti-titin monoclonal antibody.

Techniques: Western Blot, Expressing, Muscles, SDS Page, Staining, Incubation, Injection

Journal: The Journal of Cell Biology

Article Title: Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

doi: 10.1083/jcb.200107037

Figure Lengend Snippet: Immunofluorescence localization of smitin and titin in cryosections of chicken cardiac striated muscle. A 10-μm thick cryosection of chicken cardiac muscle was double labeled with an anti-smitin polyclonal antibody and anti–rabbit Ig secondary antibody (A, green) and the 9D10 anti-titin monoclonal antibody and anti–mouse IgM secondary antibody (B, red). A single 0.4-μm thick optical section is shown. Panel C contains a merged image with an inset displaying a region of the original image (small box) magnified 2.5× (large box). Bar, 10 μm.

Article Snippet: For double-label immunofluorescence analysis, the gizzard smooth muscle sections were incubated simultaneously in the anti-smitin polyclonal antibody and an anti-pan myosin monoclonal antibody (BAbCO MMS-456S; Innovative Products), and the cardiac muscle was incubated with the anti-smitin polyclonal antibody and the 9D10 anti-titin monoclonal antibody.

Techniques: Immunofluorescence, Labeling

Journal: The Journal of Cell Biology

Article Title: Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

doi: 10.1083/jcb.200107037

Figure Lengend Snippet: Immunofluorescence localization of smitin and myosin in cryosections of chicken gizzard smooth muscle. A cryosection of chicken gizzard muscle was double-labeled with an anti-smitin polyclonal antibody (A, green) and an anti-myosin monoclonal antibody (B, red) and imaged with laser scanning confocal microscopy. A single 0.55-μm thick optical section is shown. Panel C is a merged image with insets of two areas (blue and turquoise boxes) of the image magnified to twice the original size. One inset (blue boxes) shows what appears to be a smooth muscle myofibril with the anti-smitin label (green) distributed at regular intervals along a strand stained continuously along its length for myosin (red). The other inset (turquoise boxes) shows what appears to be a cross-sectional area of a strand containing both smitin (green) and myosin (red) in close association (yellow). The large open area in the center of the section is a cross section of a blood vessel through the smooth muscle. Bar, 10 μm.

Article Snippet: For double-label immunofluorescence analysis, the gizzard smooth muscle sections were incubated simultaneously in the anti-smitin polyclonal antibody and an anti-pan myosin monoclonal antibody (BAbCO MMS-456S; Innovative Products), and the cardiac muscle was incubated with the anti-smitin polyclonal antibody and the 9D10 anti-titin monoclonal antibody.

Techniques: Immunofluorescence, Labeling, Confocal Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

doi: 10.1083/jcb.200107037

Figure Lengend Snippet: Confocal microscopy images of smitin and myosin in vitro reconstituted coassembly structures. Mixtures of smitin and smooth muscle myosin were dialyzed into assembly buffers containing 50 mM KCl (A–C) to form coassembly structures containing minibipolar filaments of myosin, or 150 mM KCl to form coassembly structures containing sidepolar filaments of myosin (D–F). The coassembled structures were adhered to poly- l -lysine–coated coverslips and double labeled with an anti-smitin polyclonal antibody (green) and an anti-myosin monoclonal antibody (red). Merged images of A and B (C) and D and E (F) contain insets of the boxed regions that are magnified 2×. Bar, 5 μm.

Article Snippet: For double-label immunofluorescence analysis, the gizzard smooth muscle sections were incubated simultaneously in the anti-smitin polyclonal antibody and an anti-pan myosin monoclonal antibody (BAbCO MMS-456S; Innovative Products), and the cardiac muscle was incubated with the anti-smitin polyclonal antibody and the 9D10 anti-titin monoclonal antibody.

Techniques: Confocal Microscopy, In Vitro, Labeling

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: The interaction between I-band titin domains and sHSPs in GST pulldown assays. Representative Western blots demonstrating the presence or absence of an interaction of HSP27 (A) or αB-crystallin (B) with various I-band titin domains. (C) Overview of HSP27 and αB-crystallin interaction sites on I-band titin detected by this assay. Results marked by as asterisk were obtained previously . The I9-12 Ig segment likely also binds αB-crystallin . asPEVK, alternatively spliced PEVK.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Binding of exogenous HSP27 and αB-crystallin to the I-band region of isolated myofibrils. (A) The experimental design for the stretching of single myofibrils as well as images of stretched myofibrils incubated in relaxing buffer with GFP or with Cy3-conjugated secondary antibodies alone (negative controls). Representative immunofluorescence (FL) and phase-contrast (PC) images of human cardiac (B–D) and rabbit psoas myofibrils (E and F) incubated with recombinant sHSP at the SL indicated on left. Binding was visualized using anti-sHSP primary and Cy3-conjugated secondary antibodies. (B, D, and E) Exogenous HSP27 visualized by anti-HSP27 staining. (C and F) Exogenous αB-crystallin visualized by anti-αB-crystallin staining. In D, the actin filaments were extracted from human cardiac myofibrils using a Ca 2+ -independent gelsolin fragment before incubation with HSP27 and antibodies; images on the right demonstrate the efficient removal of actin, except in a narrow portion of the Z-disk. The profile plot taken along the myofibril axis compared the position of HSP27 in the I-bands (red dashed lines) with that of the remnant actin in the Z-disks after gelsolin treatment (black dashed line). Arrowheads, Z-disk. Bars, 5 µm.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Binding Assay, Isolation, Incubation, Immunofluorescence, Recombinant, Staining

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: pH-dependent aggregation of partially unfolded titin Ig constructs and protection by sHSPs. Titin fragments were denatured in 8 mol/l of urea and then diluted 1:50 in neutral (pH 7.2) or acidic (pH 6.7) Tris/HCl buffer (30 mmol/l). Aggregation was determined by measuring the absorbance at 320 nm for up to 30 min. (A) Lack of aggregation of the mostly disordered segments, N2-Bus, N2-B, and PEVK, at normal and acidic pH. (B) Varied aggregation propensity of Ig-rich constructs, I9-12 and N2-A, at normal and acidic pH. (C) Protection by αB-crystallin against aggregation of the N2-A construct in pH 6.7 buffer at different N2-A/αB-crystallin molar ratios. (D) Lack of an anti-aggregation effect of HSP27 on N2-A at pH 6.7. All data were normalized to the absorbance value of N2-A at pH 6.7 at the 30-min time point and represent means ± SD ( n = 6).

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Construct

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: The increase of passive tension in human skinned cardiomyocytes prestretched under acidic stress and protection by sHSPs. (A) The original recordings for the force response to stepwise cell stretches in relaxing buffer, first at pH 7.4 (black curves) then at pH 6.6 after a 10-min prestretch to 2.6-µm SL in the absence (blue curve) or presence (red curve) of sHSP. (B) Passive tension (F passive ) at pH 7.4 (Control) and at pH 6.6. Cells were not prestretched. (C and E) The F passive at pH 7.4 (black curve) and at acidic pH after prestretch and hold to a 2.6-µm SL in the absence (blue curve) or presence (red curve) of sHSPs (0.1 mg/ml). Insets in C and E show the reduction of the F passive (at 2.2-µm SL) in pH 6.6 buffer using sHSP concentrations of 0.01, 0.1, or 1 mg/ml. (D and F) The F passive at pH 7.4 (black curve) and after a prestretch and hold to a 2.6-µm SL in the absence (blue curve) or presence (red curve) of sHSP (0.1 mg/ml) at normal pH 7.4. HSP27 (C and D) and αB-crystallin (E and F) were used. Data represent means ± SEM ( n = 5–10 cells, derived from two human donor hearts); curves represent regression models of order 3; *, P < 0.05, prestretch (pH 6.6) versus prestretch + sHSP (pH 6.6).

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: HSP27 and αB-crystallin localize to the I-bands in adult but not fetal rat cardiomyocyte cultures. (A) Schematics of the titin isoform sizes in fetal and adult rat cardiac sarcomeres. (B) Typical immunofluorescence images of fetal rat cardiomyocytes staining for HSP25/27 (secondary antibody: FITC-conjugated IgG) or endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG; left) and counterstaining for A-band marker myosin-binding protein C (MyBP-C) (secondary antibody: Cy3-conjugated IgG) and Z-disk marker α-actinin (secondary antibody: Cy3-conjugated IgG), respectively. (C) Typical immunofluorescence images of adult rat cardiomyocytes staining for endogenous HSP25/27 (secondary antibody: Cy3-conjugated IgG) or endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG) and counterstaining for A-band marker MyBP-C (secondary antibody: FITC-conjugated IgG) and Z-disk marker α-actinin (secondary antibody: Cy3-conjugated IgG), respectively. (insets) Higher-power images of the regions of interest indicated in the main panels. Arrows point to a doublet indicative of I-band binding of the sHSPs. Right panels show merged images. Rat HSP25 is homologous to human HSP27. Bars: (main) 10 µm; (insets) 2 µm.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Immunofluorescence, Staining, Marker, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Binding of sHSPs to the elastic I-bands in human dystrophic skeletal muscle. (A and B) Representative immunofluorescence images of sectioned muscle biopsies comparing tissue from healthy subjects with that from LGMD2A patients. (A) Immunostaining for endogenous HSP27 (secondary antibody: FITC-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). (B) Immunostaining for endogenous αB-crystallin (secondary antibody: Cy3-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: FITC-conjugated IgG; middle). Right panels show merged images. (insets) Higher-power images of sections differing from those shown in the respective main panels. Bars, 5 µm. (C) Relative I-band positions of endogenous sHSPs and PEVK at different SLs in myocytes from LGMD2A patients (left). Each data point represents an epitope–epitope (E-E) distance value measured across the Z-disk. Regression curves are of order 2. The right panel illustrates the range of sHSP binding locations on titin.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Binding Assay, Immunofluorescence, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Binding of sHSPs to the elastic I-bands in failing human heart. (A and B) Representative immunofluorescence images of sectioned myocardial tissue from control donor hearts and failing hearts with DCM. (A) Immunostaining for endogenous HSP27 (secondary antibody: FITC-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). (B) Immunostaining for endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). Right panels show merged images. (insets) Higher-power images from sections differing from those shown in the respective main panels. Bars, 5 µm. (C) Relative I-band positions of endogenous sHSP and PEVK at different SLs in DCM cardiomyocytes (left). Each data point represents an epitope–epitope (E-E) distance value measured across the Z-disk. Regression curves are of order 2. The right panel illustrates the range of sHSP binding locations on titin.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Binding Assay, Immunofluorescence, Immunostaining

Journal: Acta Neuropathologica Communications

Article Title: Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant

doi: 10.1186/s40478-023-01539-4

Figure Lengend Snippet: RNA and protein studies. A , B RT-PCR agarose gel electrophoresis using two different pairs primers showing a detectable band above the expected one, indicated by a white star. C Schematic representation of the retained introns in P1 versus CTR. D Muscle homogenate gel electrophoresis of P1 and a control. The molecular masses of the protein are shown on the left. The Titin degradation products, nebulin and myosin are also shown. MHC was used as a loading control. Western blot analysis of patient muscle samples using different antibodies detecting different titin domains. Anti-titin antibodies: N2A isoform (Mouse N2A T5650 USbiological), A-band (Rabbit A169 #11-96 Myomedix), Z-disk (Rabbit Z1Z2 TTN-1 Miomedix), PEVK region (Mouse PEVK 9D10 DSHB). Titin degradation products and their predicted molecular weight are indicated on the left. The data are presented as mean ± SEM

Article Snippet: Anti-titin antibodies: N2A isoform (Mouse N2A T5650 USbiological), A-band (Rabbit A169 #11-96 Myomedix), Z-disk (Rabbit Z1Z2 TTN-1 Miomedix), PEVK region (Mouse PEVK 9D10 DSHB).

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Control, Western Blot, Molecular Weight

Journal: Acta Neuropathologica Communications

Article Title: Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant

doi: 10.1186/s40478-023-01539-4

Figure Lengend Snippet: Schematic representation of the N2A human isoform of titin. Yellow boxes represent the normally spliced exons, the white insertions represent the skipped exons. The pink box, represents all the exons potentially expressed in the PEVK region of the isoform, also known as metatranscript-only exons. The inclusion rate of exon 170 in the N2A human isoforms is 4%

Article Snippet: Anti-titin antibodies: N2A isoform (Mouse N2A T5650 USbiological), A-band (Rabbit A169 #11-96 Myomedix), Z-disk (Rabbit Z1Z2 TTN-1 Miomedix), PEVK region (Mouse PEVK 9D10 DSHB).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: The interaction between I-band titin domains and sHSPs in GST pulldown assays. Representative Western blots demonstrating the presence or absence of an interaction of HSP27 (A) or αB-crystallin (B) with various I-band titin domains. (C) Overview of HSP27 and αB-crystallin interaction sites on I-band titin detected by this assay. Results marked by as asterisk were obtained previously . The I9-12 Ig segment likely also binds αB-crystallin . asPEVK, alternatively spliced PEVK.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Binding of exogenous HSP27 and αB-crystallin to the I-band region of isolated myofibrils. (A) The experimental design for the stretching of single myofibrils as well as images of stretched myofibrils incubated in relaxing buffer with GFP or with Cy3-conjugated secondary antibodies alone (negative controls). Representative immunofluorescence (FL) and phase-contrast (PC) images of human cardiac (B–D) and rabbit psoas myofibrils (E and F) incubated with recombinant sHSP at the SL indicated on left. Binding was visualized using anti-sHSP primary and Cy3-conjugated secondary antibodies. (B, D, and E) Exogenous HSP27 visualized by anti-HSP27 staining. (C and F) Exogenous αB-crystallin visualized by anti-αB-crystallin staining. In D, the actin filaments were extracted from human cardiac myofibrils using a Ca 2+ -independent gelsolin fragment before incubation with HSP27 and antibodies; images on the right demonstrate the efficient removal of actin, except in a narrow portion of the Z-disk. The profile plot taken along the myofibril axis compared the position of HSP27 in the I-bands (red dashed lines) with that of the remnant actin in the Z-disks after gelsolin treatment (black dashed line). Arrowheads, Z-disk. Bars, 5 µm.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Binding Assay, Isolation, Incubation, Immunofluorescence, Recombinant, Staining

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: pH-dependent aggregation of partially unfolded titin Ig constructs and protection by sHSPs. Titin fragments were denatured in 8 mol/l of urea and then diluted 1:50 in neutral (pH 7.2) or acidic (pH 6.7) Tris/HCl buffer (30 mmol/l). Aggregation was determined by measuring the absorbance at 320 nm for up to 30 min. (A) Lack of aggregation of the mostly disordered segments, N2-Bus, N2-B, and PEVK, at normal and acidic pH. (B) Varied aggregation propensity of Ig-rich constructs, I9-12 and N2-A, at normal and acidic pH. (C) Protection by αB-crystallin against aggregation of the N2-A construct in pH 6.7 buffer at different N2-A/αB-crystallin molar ratios. (D) Lack of an anti-aggregation effect of HSP27 on N2-A at pH 6.7. All data were normalized to the absorbance value of N2-A at pH 6.7 at the 30-min time point and represent means ± SD ( n = 6).

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Construct

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: The increase of passive tension in human skinned cardiomyocytes prestretched under acidic stress and protection by sHSPs. (A) The original recordings for the force response to stepwise cell stretches in relaxing buffer, first at pH 7.4 (black curves) then at pH 6.6 after a 10-min prestretch to 2.6-µm SL in the absence (blue curve) or presence (red curve) of sHSP. (B) Passive tension (F passive ) at pH 7.4 (Control) and at pH 6.6. Cells were not prestretched. (C and E) The F passive at pH 7.4 (black curve) and at acidic pH after prestretch and hold to a 2.6-µm SL in the absence (blue curve) or presence (red curve) of sHSPs (0.1 mg/ml). Insets in C and E show the reduction of the F passive (at 2.2-µm SL) in pH 6.6 buffer using sHSP concentrations of 0.01, 0.1, or 1 mg/ml. (D and F) The F passive at pH 7.4 (black curve) and after a prestretch and hold to a 2.6-µm SL in the absence (blue curve) or presence (red curve) of sHSP (0.1 mg/ml) at normal pH 7.4. HSP27 (C and D) and αB-crystallin (E and F) were used. Data represent means ± SEM ( n = 5–10 cells, derived from two human donor hearts); curves represent regression models of order 3; *, P < 0.05, prestretch (pH 6.6) versus prestretch + sHSP (pH 6.6).

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: HSP27 and αB-crystallin localize to the I-bands in adult but not fetal rat cardiomyocyte cultures. (A) Schematics of the titin isoform sizes in fetal and adult rat cardiac sarcomeres. (B) Typical immunofluorescence images of fetal rat cardiomyocytes staining for HSP25/27 (secondary antibody: FITC-conjugated IgG) or endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG; left) and counterstaining for A-band marker myosin-binding protein C (MyBP-C) (secondary antibody: Cy3-conjugated IgG) and Z-disk marker α-actinin (secondary antibody: Cy3-conjugated IgG), respectively. (C) Typical immunofluorescence images of adult rat cardiomyocytes staining for endogenous HSP25/27 (secondary antibody: Cy3-conjugated IgG) or endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG) and counterstaining for A-band marker MyBP-C (secondary antibody: FITC-conjugated IgG) and Z-disk marker α-actinin (secondary antibody: Cy3-conjugated IgG), respectively. (insets) Higher-power images of the regions of interest indicated in the main panels. Arrows point to a doublet indicative of I-band binding of the sHSPs. Right panels show merged images. Rat HSP25 is homologous to human HSP27. Bars: (main) 10 µm; (insets) 2 µm.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Immunofluorescence, Staining, Marker, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Stretching of intact rat heart triggers the translocation of αB-crystallin to the elastic I-bands of cardiac sarcomeres. Excised adult rat hearts were perfused with PBS for 30 min, either at low pressure to avoid sarcomere stretch (left) or at high pressure to obtain highly extended sarcomeres (right). Left ventricular sections were immunostained for endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG; top) and counterstained for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). The representative images for each condition are shown. Higher-power images of the region of interest indicated are shown above the main panels. Arrowheads point to the Z-disk localization of αB-crystallin and arrows to I-band localization. Bottom panels show merged images. Bars, 5 µm.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Translocation Assay

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Binding of sHSPs to the elastic I-bands in human dystrophic skeletal muscle. (A and B) Representative immunofluorescence images of sectioned muscle biopsies comparing tissue from healthy subjects with that from LGMD2A patients. (A) Immunostaining for endogenous HSP27 (secondary antibody: FITC-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). (B) Immunostaining for endogenous αB-crystallin (secondary antibody: Cy3-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: FITC-conjugated IgG; middle). Right panels show merged images. (insets) Higher-power images of sections differing from those shown in the respective main panels. Bars, 5 µm. (C) Relative I-band positions of endogenous sHSPs and PEVK at different SLs in myocytes from LGMD2A patients (left). Each data point represents an epitope–epitope (E-E) distance value measured across the Z-disk. Regression curves are of order 2. The right panel illustrates the range of sHSP binding locations on titin.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Binding Assay, Immunofluorescence, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

doi: 10.1083/jcb.201306077

Figure Lengend Snippet: Binding of sHSPs to the elastic I-bands in failing human heart. (A and B) Representative immunofluorescence images of sectioned myocardial tissue from control donor hearts and failing hearts with DCM. (A) Immunostaining for endogenous HSP27 (secondary antibody: FITC-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). (B) Immunostaining for endogenous αB-crystallin (secondary antibody: FITC-conjugated IgG; left) and counterstaining for PEVK titin (secondary antibody: Cy3-conjugated IgG; middle). Right panels show merged images. (insets) Higher-power images from sections differing from those shown in the respective main panels. Bars, 5 µm. (C) Relative I-band positions of endogenous sHSP and PEVK at different SLs in DCM cardiomyocytes (left). Each data point represents an epitope–epitope (E-E) distance value measured across the Z-disk. Regression curves are of order 2. The right panel illustrates the range of sHSP binding locations on titin.

Article Snippet: The primary antibodies used were anti-HSP27 (1:250 dilution in PBS; SMC161 5D12; StressMarq), anti-HSP27 (1:200; SR B800; MBL), anti–αB-crystallin (1:250; SMC 165 3A10; StressMarq), anti–αB-crystallin (1:400; SR 223F; MBL), anti-titin PEVK (1:200; 9D10; Hybridoma Bank), and anti-titin PEVK (1:500; custom-made, affinity-purified; Eurogentec; ).

Techniques: Binding Assay, Immunofluorescence, Immunostaining