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Proteintech dnajb2 polyclonal antibody

Schematic representation of the human <t>DNAJB2</t> gene exons, resultant DNAJB2 protein isoforms and the mutation affecting the protein region. ( a ). The figure depicts the DNAJB2 gene, highlighting two transcript variants ( NM_006736.6 and NM_001039550.2 ) corresponding to protein isoforms b and a, respectively. The figure also highlights the potential regions in the transcript and the resulting protein that may be impacted by the mutation. ( b ). The table presents key attributes of the transcripts, including the number of exons, transcript lengths, and the resulting protein isoforms. A missense mutation (c.829C>T, p. Arg277Trp) is identified in Isoform b. In contrast, Isoform a contains an intronic variant (c.823+6C>T). The respective protein products exhibit molecular weights of 35.6 kDa for Isoform b and 30.6 kDa for Isoform a, with distinct cellular localizations. Isoform b is anchored to the cytoplasmic face of the endoplasmic reticulum, while Isoform a localizes in both the nucleus and cytoplasm. The predominant expression of Isoform b is indicated, which may be relevant to the pathogenic mechanism in this case.

Dnajb2 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dnajb2 polyclonal antibody - by Bioz Stars, 2026-08

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1) Product Images from "Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention"

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

Journal: Neurology International

doi: 10.3390/neurolint17050073

Schematic representation of the human DNAJB2 gene exons, resultant DNAJB2 protein isoforms and the mutation affecting the protein region. ( a ). The figure depicts the DNAJB2 gene, highlighting two transcript variants ( NM_006736.6 and NM_001039550.2 ) corresponding to protein isoforms b and a, respectively. The figure also highlights the potential regions in the transcript and the resulting protein that may be impacted by the mutation. ( b ). The table presents key attributes of the transcripts, including the number of exons, transcript lengths, and the resulting protein isoforms. A missense mutation (c.829C>T, p. Arg277Trp) is identified in Isoform b. In contrast, Isoform a contains an intronic variant (c.823+6C>T). The respective protein products exhibit molecular weights of 35.6 kDa for Isoform b and 30.6 kDa for Isoform a, with distinct cellular localizations. Isoform b is anchored to the cytoplasmic face of the endoplasmic reticulum, while Isoform a localizes in both the nucleus and cytoplasm. The predominant expression of Isoform b is indicated, which may be relevant to the pathogenic mechanism in this case.

Figure Legend Snippet: Schematic representation of the human DNAJB2 gene exons, resultant DNAJB2 protein isoforms and the mutation affecting the protein region. ( a ). The figure depicts the DNAJB2 gene, highlighting two transcript variants ( NM_006736.6 and NM_001039550.2 ) corresponding to protein isoforms b and a, respectively. The figure also highlights the potential regions in the transcript and the resulting protein that may be impacted by the mutation. ( b ). The table presents key attributes of the transcripts, including the number of exons, transcript lengths, and the resulting protein isoforms. A missense mutation (c.829C>T, p. Arg277Trp) is identified in Isoform b. In contrast, Isoform a contains an intronic variant (c.823+6C>T). The respective protein products exhibit molecular weights of 35.6 kDa for Isoform b and 30.6 kDa for Isoform a, with distinct cellular localizations. Isoform b is anchored to the cytoplasmic face of the endoplasmic reticulum, while Isoform a localizes in both the nucleus and cytoplasm. The predominant expression of Isoform b is indicated, which may be relevant to the pathogenic mechanism in this case.

Techniques Used: Mutagenesis, Variant Assay, Expressing

Overview of the heat shock response pathway and the potential effects of the c.823+6C>T mutation on the capacity of DNAJB2 to manage cellular stress mechanisms. 1. Stressors such as heat, oxidative stress, etc., can trigger the heat shock response pathway. 2. The cell senses stress and initiates the heat shock response. 3. Release of Heat Shock Factors (HSFs) from their inactive state in the cytoplasm 4. Activated HSFs translocate to the nucleus. 5. HSFs bind to Heat Shock Elements (HSE), initiating the transcription of heat shock proteins (HSPs). 6. HSPs, including DNAJB2 (HSP40), are synthesized to refold or degrade damaged proteins. 7. DNAJB2 works with HSP70 to maintain protein homeostasis by refolding proteins or directing damaged ones for degradation.

Figure Legend Snippet: Overview of the heat shock response pathway and the potential effects of the c.823+6C>T mutation on the capacity of DNAJB2 to manage cellular stress mechanisms. 1. Stressors such as heat, oxidative stress, etc., can trigger the heat shock response pathway. 2. The cell senses stress and initiates the heat shock response. 3. Release of Heat Shock Factors (HSFs) from their inactive state in the cytoplasm 4. Activated HSFs translocate to the nucleus. 5. HSFs bind to Heat Shock Elements (HSE), initiating the transcription of heat shock proteins (HSPs). 6. HSPs, including DNAJB2 (HSP40), are synthesized to refold or degrade damaged proteins. 7. DNAJB2 works with HSP70 to maintain protein homeostasis by refolding proteins or directing damaged ones for degradation.

Techniques Used: Mutagenesis, Synthesized

Quantitative PCR analysis of HSJ1a and HSJ1b gene expression at various time points following a one-hour heat shock. ( a ). Schematic representation of the experimental setup showing the timing of heat shock and subsequent sampling for gene expression analysis. Gene expression levels in all the groups were normalized to their respective expression of housekeeping gene GAPDH. ( b ). Bar graph depicting the basal expression of the HSJ1a gene in control fibroblasts (JWAC-25 and JWAC-83) relative to its expression in DNAJB2-mutated patient fibroblasts (JWAC-39). ( c ). Bar graph illustrating the expression of the HSJ1a gene in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 3 h and 24 h post one-hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( d ). Bar graph displaying the expression of the HSJ1a gene expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 3 h and 24 h post one-hour heat shock, with baseline and treatment conditions for all lines normalized to the baseline expression of the mutant fibroblasts. For ( e – g ), cells were treated as in b–d but were now evaluated for HSJ1b gene expression. Values are expressed as mean ± SEM. Statistical significance was assessed using the Friedman test with uncorrected Dunn’s test to compare the baseline levels, whereas repeated measures two-way ANOVA with uncorrected Fisher’s LSD was applied for comparing the gene expression of HSJ1a and HSJ1b at various time points following a one-hour heat shock (n = 5). Results were considered statistically significant at p * < 0.05, ** < 0.01, C-1 = Control 1, C-2 = Control 2, mut. = mutant. Note: ( b , e ) are evaluating the basal levels without normalization; ( c , f ) are looking at the response of the individual line over time, and ( d , g ) are comparing the response at that given time.

Figure Legend Snippet: Quantitative PCR analysis of HSJ1a and HSJ1b gene expression at various time points following a one-hour heat shock. ( a ). Schematic representation of the experimental setup showing the timing of heat shock and subsequent sampling for gene expression analysis. Gene expression levels in all the groups were normalized to their respective expression of housekeeping gene GAPDH. ( b ). Bar graph depicting the basal expression of the HSJ1a gene in control fibroblasts (JWAC-25 and JWAC-83) relative to its expression in DNAJB2-mutated patient fibroblasts (JWAC-39). ( c ). Bar graph illustrating the expression of the HSJ1a gene in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 3 h and 24 h post one-hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( d ). Bar graph displaying the expression of the HSJ1a gene expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 3 h and 24 h post one-hour heat shock, with baseline and treatment conditions for all lines normalized to the baseline expression of the mutant fibroblasts. For ( e – g ), cells were treated as in b–d but were now evaluated for HSJ1b gene expression. Values are expressed as mean ± SEM. Statistical significance was assessed using the Friedman test with uncorrected Dunn’s test to compare the baseline levels, whereas repeated measures two-way ANOVA with uncorrected Fisher’s LSD was applied for comparing the gene expression of HSJ1a and HSJ1b at various time points following a one-hour heat shock (n = 5). Results were considered statistically significant at p * < 0.05, ** < 0.01, C-1 = Control 1, C-2 = Control 2, mut. = mutant. Note: ( b , e ) are evaluating the basal levels without normalization; ( c , f ) are looking at the response of the individual line over time, and ( d , g ) are comparing the response at that given time.

Techniques Used: Real-time Polymerase Chain Reaction, Gene Expression, Sampling, Expressing, Control, Mutagenesis

Western blot analysis of DNAJB2 protein levels at various time points following a one-hour heat shock. ( a ). Experimental layout illustrating the timing of heat shock and subsequent sampling for protein expression analysis of HSJ1b. The expression of all the proteins were normalized to their respective total protein. ( b ). Representative immunoblot image displaying the expression of HSJ1a (36 kDa) and HSJ1b (40 kDa) bands in control (JWAC-25 and JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock. The lower panel shows their respective total protein stains confirming equal loading of proteins (20 µg/lane). ( c ). Densitometric analysis of the basal expression of HSJ1b protein in the control fibroblasts (JWAC-25 and JWAC−83) relative to those expressed in mut. DNAJB2 patient fibroblasts (JWAC-39). ( d ). Densitometric analysis of HSJ1b protein expression in control (JWAC−25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one−hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( e ). Densitometric analysis of HSJ1b protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to the basal expression in mut. DNAJB2 patient fibroblasts. ( f ). Densitometric analysis of HSJ1a protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( g ). Densitometric analysis of HSJ1a protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to the basal expression in mut. DNAJB2 patient fibroblasts. Values are expressed as mean ± SEM. For statistics, Friedman test with uncorrected Dunn’s test was used to compare the baseline levels whereas a mixed-effects model with uncorrected Fisher’s LSD was used to compare the expression of DNAJB2 at various time points following a one-hour heat shock. Results were considered statistically significant at p * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (The sample size for DNAJB2 expression is n = 6, except at the 8 h time point (n = 3). C-1 = Control 1, C-2 = Control 2, mut. = mutant. Note: ( c ) is evaluating the basal levels without normalization; ( d , f ) is looking at the response of the individual line over time; and ( e , g ) are comparing the response at that given time.

Figure Legend Snippet: Western blot analysis of DNAJB2 protein levels at various time points following a one-hour heat shock. ( a ). Experimental layout illustrating the timing of heat shock and subsequent sampling for protein expression analysis of HSJ1b. The expression of all the proteins were normalized to their respective total protein. ( b ). Representative immunoblot image displaying the expression of HSJ1a (36 kDa) and HSJ1b (40 kDa) bands in control (JWAC-25 and JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock. The lower panel shows their respective total protein stains confirming equal loading of proteins (20 µg/lane). ( c ). Densitometric analysis of the basal expression of HSJ1b protein in the control fibroblasts (JWAC-25 and JWAC−83) relative to those expressed in mut. DNAJB2 patient fibroblasts (JWAC-39). ( d ). Densitometric analysis of HSJ1b protein expression in control (JWAC−25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one−hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( e ). Densitometric analysis of HSJ1b protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to the basal expression in mut. DNAJB2 patient fibroblasts. ( f ). Densitometric analysis of HSJ1a protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( g ). Densitometric analysis of HSJ1a protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to the basal expression in mut. DNAJB2 patient fibroblasts. Values are expressed as mean ± SEM. For statistics, Friedman test with uncorrected Dunn’s test was used to compare the baseline levels whereas a mixed-effects model with uncorrected Fisher’s LSD was used to compare the expression of DNAJB2 at various time points following a one-hour heat shock. Results were considered statistically significant at p * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (The sample size for DNAJB2 expression is n = 6, except at the 8 h time point (n = 3). C-1 = Control 1, C-2 = Control 2, mut. = mutant. Note: ( c ) is evaluating the basal levels without normalization; ( d , f ) is looking at the response of the individual line over time; and ( e , g ) are comparing the response at that given time.

Techniques Used: Western Blot, Sampling, Expressing, Control, Mutagenesis

Cell viability assay of control and mut. DNAJB2 fibroblasts post one-hour of heat shock. Fibroblasts were grown in two plates and one of them was subjected to one-hour of heat shock (heat shock plate). CellTox Green Dye was added one day before the heat shock treatment. Fluorescence was measured at 0, 1, 4, 8, 12, 16, 20, and 24 h post heat shock treatment. Percentage of cell death in each line were calculated relative to those caused by 1% Triton X-100 (positive control) set to 100%. Cell death in the control fibroblasts JWAC-25 (Purple), JWAC-83 (Brown) and JWAC-39 (green) in the no heat shock plate and JWAC-25 (black), JWAC-83 (Pink) and JWAC-39 (orange) in the heat shock plate were plotted at different time points post heat shock. Values are expressed as mean ± SD (n = 4, technical replicates). Note: There was an initial dip in the fluorescence intensity immediately following heat shock in the treated groups. This transient dip is attributed to the instability of the CellTox™ Green dye at elevated temperatures, such as 42 °C. The dye’s fluorescence stabilized after approximately 3 h. Therefore, the negative fluorescence values recorded during this period are technical artifacts and should not be interpreted as indicators of cell viability.

Figure Legend Snippet: Cell viability assay of control and mut. DNAJB2 fibroblasts post one-hour of heat shock. Fibroblasts were grown in two plates and one of them was subjected to one-hour of heat shock (heat shock plate). CellTox Green Dye was added one day before the heat shock treatment. Fluorescence was measured at 0, 1, 4, 8, 12, 16, 20, and 24 h post heat shock treatment. Percentage of cell death in each line were calculated relative to those caused by 1% Triton X-100 (positive control) set to 100%. Cell death in the control fibroblasts JWAC-25 (Purple), JWAC-83 (Brown) and JWAC-39 (green) in the no heat shock plate and JWAC-25 (black), JWAC-83 (Pink) and JWAC-39 (orange) in the heat shock plate were plotted at different time points post heat shock. Values are expressed as mean ± SD (n = 4, technical replicates). Note: There was an initial dip in the fluorescence intensity immediately following heat shock in the treated groups. This transient dip is attributed to the instability of the CellTox™ Green dye at elevated temperatures, such as 42 °C. The dye’s fluorescence stabilized after approximately 3 h. Therefore, the negative fluorescence values recorded during this period are technical artifacts and should not be interpreted as indicators of cell viability.

Techniques Used: Viability Assay, Control, CellTox Assay, Fluorescence, Positive Control

Effect of Cystamine pretreatment on the levels of DNAJB2 (HSP40). ( a ). Experimental layout illustrating the time of introduction of Cystamine, and a washout period maintained to study its effect on the expression of HSJ1b protein levels. The expression of HSJ1b in different groups were normalized to their respective total protein. ( b ). Representative immunoblot image showing the expression of HSJ1a (36 kDa) and HSJ1b (40 kDa) bands in the controls (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts, detected with anti-DNAJB2 antibody across different exposure times and washout periods of Cystamine (150 µM). The lower panel represents their respective total protein stains, confirming comparable loading of proteins (20 µg/lane). ( c ). Densitometric analysis of the expression of HSJ1b isoform protein in control (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts across different exposure times and washout periods of Cystamine, normalized to the basal expression of the protein in their respective untreated fibroblasts. ( d ). Densitometric analysis of the expression of HSJ1a isoform proteins in control (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts across different exposure times and washout periods of Cystamine, normalized to the basal expression of the protein in their respective untreated fibroblasts. Values are expressed as mean ± SEM. For statistics, repeated measures two-way ANOVA with uncorrected Fisher’s LSD were used. Results were considered statistically significant at p * < 0.05 (n = 3 for DNAJB2 expression). (Stab. = Stabilization).

Figure Legend Snippet: Effect of Cystamine pretreatment on the levels of DNAJB2 (HSP40). ( a ). Experimental layout illustrating the time of introduction of Cystamine, and a washout period maintained to study its effect on the expression of HSJ1b protein levels. The expression of HSJ1b in different groups were normalized to their respective total protein. ( b ). Representative immunoblot image showing the expression of HSJ1a (36 kDa) and HSJ1b (40 kDa) bands in the controls (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts, detected with anti-DNAJB2 antibody across different exposure times and washout periods of Cystamine (150 µM). The lower panel represents their respective total protein stains, confirming comparable loading of proteins (20 µg/lane). ( c ). Densitometric analysis of the expression of HSJ1b isoform protein in control (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts across different exposure times and washout periods of Cystamine, normalized to the basal expression of the protein in their respective untreated fibroblasts. ( d ). Densitometric analysis of the expression of HSJ1a isoform proteins in control (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts across different exposure times and washout periods of Cystamine, normalized to the basal expression of the protein in their respective untreated fibroblasts. Values are expressed as mean ± SEM. For statistics, repeated measures two-way ANOVA with uncorrected Fisher’s LSD were used. Results were considered statistically significant at p * < 0.05 (n = 3 for DNAJB2 expression). (Stab. = Stabilization).

Techniques Used: Expressing, Western Blot, Control

Image Search Results

Journal: Neurology International

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

doi: 10.3390/neurolint17050073

Figure Lengend Snippet: Schematic representation of the human DNAJB2 gene exons, resultant DNAJB2 protein isoforms and the mutation affecting the protein region. ( a ). The figure depicts the DNAJB2 gene, highlighting two transcript variants ( NM_006736.6 and NM_001039550.2 ) corresponding to protein isoforms b and a, respectively. The figure also highlights the potential regions in the transcript and the resulting protein that may be impacted by the mutation. ( b ). The table presents key attributes of the transcripts, including the number of exons, transcript lengths, and the resulting protein isoforms. A missense mutation (c.829C>T, p. Arg277Trp) is identified in Isoform b. In contrast, Isoform a contains an intronic variant (c.823+6C>T). The respective protein products exhibit molecular weights of 35.6 kDa for Isoform b and 30.6 kDa for Isoform a, with distinct cellular localizations. Isoform b is anchored to the cytoplasmic face of the endoplasmic reticulum, while Isoform a localizes in both the nucleus and cytoplasm. The predominant expression of Isoform b is indicated, which may be relevant to the pathogenic mechanism in this case.

Article Snippet: The blots were then probed with the following primary antibodies: DNAJB2 Polyclonal antibody (Proteintech, Rosemont, IL, USA, Catalog: 10838-1-AP, RRID: AB_2277491).

Techniques: Mutagenesis, Variant Assay, Expressing

Journal: Neurology International

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

doi: 10.3390/neurolint17050073

Figure Lengend Snippet: Overview of the heat shock response pathway and the potential effects of the c.823+6C>T mutation on the capacity of DNAJB2 to manage cellular stress mechanisms. 1. Stressors such as heat, oxidative stress, etc., can trigger the heat shock response pathway. 2. The cell senses stress and initiates the heat shock response. 3. Release of Heat Shock Factors (HSFs) from their inactive state in the cytoplasm 4. Activated HSFs translocate to the nucleus. 5. HSFs bind to Heat Shock Elements (HSE), initiating the transcription of heat shock proteins (HSPs). 6. HSPs, including DNAJB2 (HSP40), are synthesized to refold or degrade damaged proteins. 7. DNAJB2 works with HSP70 to maintain protein homeostasis by refolding proteins or directing damaged ones for degradation.

Article Snippet: The blots were then probed with the following primary antibodies: DNAJB2 Polyclonal antibody (Proteintech, Rosemont, IL, USA, Catalog: 10838-1-AP, RRID: AB_2277491).

Techniques: Mutagenesis, Synthesized

Journal: Neurology International

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

doi: 10.3390/neurolint17050073

Figure Lengend Snippet: Quantitative PCR analysis of HSJ1a and HSJ1b gene expression at various time points following a one-hour heat shock. ( a ). Schematic representation of the experimental setup showing the timing of heat shock and subsequent sampling for gene expression analysis. Gene expression levels in all the groups were normalized to their respective expression of housekeeping gene GAPDH. ( b ). Bar graph depicting the basal expression of the HSJ1a gene in control fibroblasts (JWAC-25 and JWAC-83) relative to its expression in DNAJB2-mutated patient fibroblasts (JWAC-39). ( c ). Bar graph illustrating the expression of the HSJ1a gene in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 3 h and 24 h post one-hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( d ). Bar graph displaying the expression of the HSJ1a gene expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 3 h and 24 h post one-hour heat shock, with baseline and treatment conditions for all lines normalized to the baseline expression of the mutant fibroblasts. For ( e – g ), cells were treated as in b–d but were now evaluated for HSJ1b gene expression. Values are expressed as mean ± SEM. Statistical significance was assessed using the Friedman test with uncorrected Dunn’s test to compare the baseline levels, whereas repeated measures two-way ANOVA with uncorrected Fisher’s LSD was applied for comparing the gene expression of HSJ1a and HSJ1b at various time points following a one-hour heat shock (n = 5). Results were considered statistically significant at p * < 0.05, ** < 0.01, C-1 = Control 1, C-2 = Control 2, mut. = mutant. Note: ( b , e ) are evaluating the basal levels without normalization; ( c , f ) are looking at the response of the individual line over time, and ( d , g ) are comparing the response at that given time.

Article Snippet: The blots were then probed with the following primary antibodies: DNAJB2 Polyclonal antibody (Proteintech, Rosemont, IL, USA, Catalog: 10838-1-AP, RRID: AB_2277491).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Sampling, Expressing, Control, Mutagenesis

Journal: Neurology International

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

doi: 10.3390/neurolint17050073

Figure Lengend Snippet: Western blot analysis of DNAJB2 protein levels at various time points following a one-hour heat shock. ( a ). Experimental layout illustrating the timing of heat shock and subsequent sampling for protein expression analysis of HSJ1b. The expression of all the proteins were normalized to their respective total protein. ( b ). Representative immunoblot image displaying the expression of HSJ1a (36 kDa) and HSJ1b (40 kDa) bands in control (JWAC-25 and JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock. The lower panel shows their respective total protein stains confirming equal loading of proteins (20 µg/lane). ( c ). Densitometric analysis of the basal expression of HSJ1b protein in the control fibroblasts (JWAC-25 and JWAC−83) relative to those expressed in mut. DNAJB2 patient fibroblasts (JWAC-39). ( d ). Densitometric analysis of HSJ1b protein expression in control (JWAC−25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one−hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( e ). Densitometric analysis of HSJ1b protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to the basal expression in mut. DNAJB2 patient fibroblasts. ( f ). Densitometric analysis of HSJ1a protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to their respective baseline expression in non-heat-shocked fibroblasts. ( g ). Densitometric analysis of HSJ1a protein expression in control (JWAC-25 and JWAC-83) and mut. DNAJB2 fibroblasts at 0, 3, 8, and 24 h post one-hour heat shock, normalized to the basal expression in mut. DNAJB2 patient fibroblasts. Values are expressed as mean ± SEM. For statistics, Friedman test with uncorrected Dunn’s test was used to compare the baseline levels whereas a mixed-effects model with uncorrected Fisher’s LSD was used to compare the expression of DNAJB2 at various time points following a one-hour heat shock. Results were considered statistically significant at p * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (The sample size for DNAJB2 expression is n = 6, except at the 8 h time point (n = 3). C-1 = Control 1, C-2 = Control 2, mut. = mutant. Note: ( c ) is evaluating the basal levels without normalization; ( d , f ) is looking at the response of the individual line over time; and ( e , g ) are comparing the response at that given time.

Article Snippet: The blots were then probed with the following primary antibodies: DNAJB2 Polyclonal antibody (Proteintech, Rosemont, IL, USA, Catalog: 10838-1-AP, RRID: AB_2277491).

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

Journal: Neurology International

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

doi: 10.3390/neurolint17050073

Figure Lengend Snippet: Cell viability assay of control and mut. DNAJB2 fibroblasts post one-hour of heat shock. Fibroblasts were grown in two plates and one of them was subjected to one-hour of heat shock (heat shock plate). CellTox Green Dye was added one day before the heat shock treatment. Fluorescence was measured at 0, 1, 4, 8, 12, 16, 20, and 24 h post heat shock treatment. Percentage of cell death in each line were calculated relative to those caused by 1% Triton X-100 (positive control) set to 100%. Cell death in the control fibroblasts JWAC-25 (Purple), JWAC-83 (Brown) and JWAC-39 (green) in the no heat shock plate and JWAC-25 (black), JWAC-83 (Pink) and JWAC-39 (orange) in the heat shock plate were plotted at different time points post heat shock. Values are expressed as mean ± SD (n = 4, technical replicates). Note: There was an initial dip in the fluorescence intensity immediately following heat shock in the treated groups. This transient dip is attributed to the instability of the CellTox™ Green dye at elevated temperatures, such as 42 °C. The dye’s fluorescence stabilized after approximately 3 h. Therefore, the negative fluorescence values recorded during this period are technical artifacts and should not be interpreted as indicators of cell viability.

Article Snippet: The blots were then probed with the following primary antibodies: DNAJB2 Polyclonal antibody (Proteintech, Rosemont, IL, USA, Catalog: 10838-1-AP, RRID: AB_2277491).

Techniques: Viability Assay, Control, CellTox Assay, Fluorescence, Positive Control

Journal: Neurology International

Article Title: Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention

doi: 10.3390/neurolint17050073

Figure Lengend Snippet: Effect of Cystamine pretreatment on the levels of DNAJB2 (HSP40). ( a ). Experimental layout illustrating the time of introduction of Cystamine, and a washout period maintained to study its effect on the expression of HSJ1b protein levels. The expression of HSJ1b in different groups were normalized to their respective total protein. ( b ). Representative immunoblot image showing the expression of HSJ1a (36 kDa) and HSJ1b (40 kDa) bands in the controls (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts, detected with anti-DNAJB2 antibody across different exposure times and washout periods of Cystamine (150 µM). The lower panel represents their respective total protein stains, confirming comparable loading of proteins (20 µg/lane). ( c ). Densitometric analysis of the expression of HSJ1b isoform protein in control (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts across different exposure times and washout periods of Cystamine, normalized to the basal expression of the protein in their respective untreated fibroblasts. ( d ). Densitometric analysis of the expression of HSJ1a isoform proteins in control (JWAC-83) and mut. DNAJB2 (JWAC-39) fibroblasts across different exposure times and washout periods of Cystamine, normalized to the basal expression of the protein in their respective untreated fibroblasts. Values are expressed as mean ± SEM. For statistics, repeated measures two-way ANOVA with uncorrected Fisher’s LSD were used. Results were considered statistically significant at p * < 0.05 (n = 3 for DNAJB2 expression). (Stab. = Stabilization).

Article Snippet: The blots were then probed with the following primary antibodies: DNAJB2 Polyclonal antibody (Proteintech, Rosemont, IL, USA, Catalog: 10838-1-AP, RRID: AB_2277491).

Techniques: Expressing, Western Blot, Control

Figure 1. Class A JDPs prevent misfolding and aggregation of mutant p53 (A–C) Aggregation of R249S and R282W destabilized p53 mutants either alone (black) or upon addition of 2-fold molar excess of DNAJA1 (maroon), DNAJA2 (red), DNAJB1 (blue), DNAJB4 (purple), DNAJB2 (teal), DNAJB6 (green), DNAJC7 (yellow), or DNAJC8 (brown), monitored by light scattering. Data are means (n = 3). (D) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 WT (blue), R249S (pink), or R282W (orange), measured at 37C. R249S and R282W p53 mutants bind DNAJA2 with 4.4 ± 0.2 and 1.2 ± 0.1 mM afﬁnity, while only a weak binding is detected for WT p53. Data are means ± SEM (n = 3). (E) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 WT at increasing temperatures (25C–44C; light to dark blue). DNAJA2 afﬁnity for WT p53 increased with temperature, from 70 mM at 37C to 10.4 mM at 40C and 4.5 mM at 44C. Data are means ± SEM (n = 3). (F) Immunoﬂuorescence staining of destabilized R249S (top) or R282W (bottom) p53 mutant overexpressed in the p53 null SaOS2 cell lines shows distinct accumulation of p53 in cytoplasmic foci, corresponding to aggregates.20 Co-expression of V5-DNAJA2, but not V5-DNAJB1, results in a signiﬁcant increase in diffuse cytoplasmic staining of mutant p53. EV, empty vector. The crop images are the overlay of p53 signal with DAPI. Scale bars: 10 mm. (G) Quantiﬁcation of cytoplasmic foci in cells overexpressing p53 WT, R249S, or R282W with and without DNAJA2 co-expression (representative images in Figures 1F and S1F). Data represent mean values ± SD (n = 3). ***p < 0.001 (Student’s t test). See also Figure S1.

Journal: Molecular cell

Article Title: A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

doi: 10.1016/j.molcel.2024.02.018

Figure Lengend Snippet: Figure 1. Class A JDPs prevent misfolding and aggregation of mutant p53 (A–C) Aggregation of R249S and R282W destabilized p53 mutants either alone (black) or upon addition of 2-fold molar excess of DNAJA1 (maroon), DNAJA2 (red), DNAJB1 (blue), DNAJB4 (purple), DNAJB2 (teal), DNAJB6 (green), DNAJC7 (yellow), or DNAJC8 (brown), monitored by light scattering. Data are means (n = 3). (D) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 WT (blue), R249S (pink), or R282W (orange), measured at 37C. R249S and R282W p53 mutants bind DNAJA2 with 4.4 ± 0.2 and 1.2 ± 0.1 mM afﬁnity, while only a weak binding is detected for WT p53. Data are means ± SEM (n = 3). (E) Fluorescence anisotropy binding assays of Alexa Fluor 488-labeled DNAJA2, titrated with increasing concentrations of p53 WT at increasing temperatures (25C–44C; light to dark blue). DNAJA2 afﬁnity for WT p53 increased with temperature, from 70 mM at 37C to 10.4 mM at 40C and 4.5 mM at 44C. Data are means ± SEM (n = 3). (F) Immunoﬂuorescence staining of destabilized R249S (top) or R282W (bottom) p53 mutant overexpressed in the p53 null SaOS2 cell lines shows distinct accumulation of p53 in cytoplasmic foci, corresponding to aggregates.20 Co-expression of V5-DNAJA2, but not V5-DNAJB1, results in a signiﬁcant increase in diffuse cytoplasmic staining of mutant p53. EV, empty vector. The crop images are the overlay of p53 signal with DAPI. Scale bars: 10 mm. (G) Quantiﬁcation of cytoplasmic foci in cells overexpressing p53 WT, R249S, or R282W with and without DNAJA2 co-expression (representative images in Figures 1F and S1F). Data represent mean values ± SD (n = 3). ***p < 0.001 (Student’s t test). See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pET29b DNAJA2mono (1-353) F337D TEV cleavable His tag This paper N/A pcDNA5 DNAJA2 with V5 tag Hageman et al.82 Addgene 19519 pcDNA5 DNAJA2 DBH D169-182 with V5 tag This paper N/A pET29b DNAJA1 TEV cleavable His tag This paper N/A pET-sumo DNAJB1 His-SUMO tag Ulp1 cleavable Nillegoda et al.61 N/A pcDNA5 DNAJB1 with V5 tag Hageman et al.82 Addgene 19522 pET29b DNAJB2 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJB4 TEV cleavable His tag Faust et al.60 N/A pET-sumo DNAJB6 His-SUMO tag Ulp1 cleavable This paper N/A pET29b DNAJC7 TEV cleavable His tag Hou et al.83 N/A pET29b DNAJC8 His-SUMO tag Ulp1 cleavable This paper N/A pET-sumo Hsp70 His-SUMO tag Ulp1 cleavable Wentink et al.54 N/A pET29b Hsp110 TEV cleavable His tag Nillegoda et al.61 N/A pET-27b p53 DBD (94-293) Kitayner et al.84 N/A pcDNA3.1 p53 Loughery et al.85 Addgene 69003 pET-27b p53 R249S DBD (94-293) Suad et al.44 N/A pcDNA3.1 p53 R249S This paper N/A pET-27b p53 R282W DBD (94-293) Degtjarik et al.46 N/A pcDNA3.1 p53 R282W This paper N/A pET-Sumo tau C291S, C322S, L243C, T373C Irwin et al.50 N/A pET SUMO Ydj1 mono (111-351) F335D His-SUMO tag Ulp1 cleavable This paper N/A Ulp1 protease Produced in house N/A Tobacco Etch Virus Protease Produced in house N/A Software and algorithms Image Studio LI-COR https://www.licor.com/bio/image- studio/resources#is5-clx ImageJ Schneider et al.86 https://imagej.nih.gov/ij/ Huygens Professional Scientific Volume Imaging https://svi.nl/Huygens-Professional NMRFAM-SPARKY Goddard and Kneller87 https://nmrfam.wisc.edu/ nmrfam-sparky-distribution/ Topspin Bruker https://www.bruker.com/en/products- and-solutions/mr/nmr-software/topspin.html NMRPipe Delaglio et al.88 https://www.ibbr.umd.edu/nmrpipe/ CcpNmrAnalysis Skinner et al.89 https://ccpn.ac.uk/software/version-2/ Prism GraphPad https://www.graphpad.com/ scientific-software/prism/ Dynamics Wyatt Technology https://www.wyatt.com/products/ software/dynamics.html ASTRA Wyatt Technology https://www.wyatt.com/products/ software/astra.html UCSF ChimeraX Pettersen et al.90 https://www.cgl.ucsf.edu/ chimerax/download.html

Techniques: Mutagenesis, Fluorescence, Binding Assay, Labeling, Staining, Expressing, Plasmid Preparation

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Published DNAJB2 mutations

Article Snippet: Primary antibodies: DNAJB2 Rb pAb (Proteintech, 10838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); TDP-43 Ms mAb (Sigma-Aldrich, WH00234, clone 2E2-D3, RRID:AB_1843869); p62 Rb pAb (Sigma-Aldrich, P0067, RRID:AB_1841064).

Techniques:

Patients and clinical findings. ( A ) Pedigree of our family. The DNAJB2 genotype (+, wild-type; ext, c.832 T > G p. * 278Glyext * 83) is shown for the family members available for genetic analysis. ( B ) Muscle MRIs of the proband (left) show diffuse neurogenic degenerative change in the soleus and gastrocnemius lateralis muscles of the lower legs but also myopathic-dystrophic fatty replacement focal changes in anterior parts of gluteus minimus and left soleus (arrows). Degenerative changes in the younger brother (right) are similar but milder with more neurogenic but also spots of myopathic replacement in the left adductor magnus (arrow) and the outer part of both peroneus longus muscles. ( C ) Gastrocnemius muscle biopsy of the proband (1 and 2 haematoxylin/eosin, 3 NADH and 4 ATPase pH 4.6) showing classical neurogenic changes such as fibre type grouping and groups of atrophic fibres together with clear myopathic changes such as rimmed vacuoles (arrowhead in 1), fibre splitting (arrowhead in 2) and heavily increased number of internalized myonuclei. Furthermore, in NADH staining , some small dark angulated fibres (black arrowhead in 3) and few moth-eaten fibres (white arrowhead in 3) are found. Scale bars 25 μm for 1 and 2, 100 μm for 3 and 4.

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Patients and clinical findings. ( A ) Pedigree of our family. The DNAJB2 genotype (+, wild-type; ext, c.832 T > G p. * 278Glyext * 83) is shown for the family members available for genetic analysis. ( B ) Muscle MRIs of the proband (left) show diffuse neurogenic degenerative change in the soleus and gastrocnemius lateralis muscles of the lower legs but also myopathic-dystrophic fatty replacement focal changes in anterior parts of gluteus minimus and left soleus (arrows). Degenerative changes in the younger brother (right) are similar but milder with more neurogenic but also spots of myopathic replacement in the left adductor magnus (arrow) and the outer part of both peroneus longus muscles. ( C ) Gastrocnemius muscle biopsy of the proband (1 and 2 haematoxylin/eosin, 3 NADH and 4 ATPase pH 4.6) showing classical neurogenic changes such as fibre type grouping and groups of atrophic fibres together with clear myopathic changes such as rimmed vacuoles (arrowhead in 1), fibre splitting (arrowhead in 2) and heavily increased number of internalized myonuclei. Furthermore, in NADH staining , some small dark angulated fibres (black arrowhead in 3) and few moth-eaten fibres (white arrowhead in 3) are found. Scale bars 25 μm for 1 and 2, 100 μm for 3 and 4.

Techniques: Muscles, Staining

The DNAJB2 mutation. ( A ) The T > G change identified in the proband affects the termination codon of the DNAJB2 transcript variant 1 (NM_001039550.2) and is predicted to cause a C-terminal extension of the DNAJB2a protein isoform (NP_001034639.1:p. * 278Glyext * 83). In variant 2 (NM_006736.6) encoding the DNAJB2b isoform, the altered nucleotide lies within the 3′ UTR and does not affect the protein product. In the diagram of the DNAJB2 transcript, the non-coding regions are shown in grey, the normal coding regions in black and the extended open reading frame caused by the mutation in magenta. Both the DNAJB2a (top) and DNAJB2b (bottom) proteins contain an N-terminal J domain (JD; orange) followed by a glycine/phenylalanine-rich region (G/F; blue), and a C-terminal domain (CTD; yellow) containing a serine-rich region (SR) and two ubiquitin-interacting motifs (UIMs). The two isoforms differ in their C-terminal parts (green), and DNAJB2b has a C-terminal geranylgeranyl moiety (GG) anchoring it to the endoplasmic reticulum. The extended DNAJB2a protein (p. * 278Glyext * 83) produced from the mutant allele has a C-terminal extension of 83 amino acids (magenta; sequence shown in the box). ( B ) RNA sequencing (RNAseq) coverage graphs covering the last exon(s) of the DNAJB2 transcripts. RNAseq of the proband (P) muscle sample showed equal expression of the wild-type and mutant alleles (arrow) and did not indicate splicing changes or altered isoform ratio compared with other samples run in the same batch (C1–5).

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: The DNAJB2 mutation. ( A ) The T > G change identified in the proband affects the termination codon of the DNAJB2 transcript variant 1 (NM_001039550.2) and is predicted to cause a C-terminal extension of the DNAJB2a protein isoform (NP_001034639.1:p. * 278Glyext * 83). In variant 2 (NM_006736.6) encoding the DNAJB2b isoform, the altered nucleotide lies within the 3′ UTR and does not affect the protein product. In the diagram of the DNAJB2 transcript, the non-coding regions are shown in grey, the normal coding regions in black and the extended open reading frame caused by the mutation in magenta. Both the DNAJB2a (top) and DNAJB2b (bottom) proteins contain an N-terminal J domain (JD; orange) followed by a glycine/phenylalanine-rich region (G/F; blue), and a C-terminal domain (CTD; yellow) containing a serine-rich region (SR) and two ubiquitin-interacting motifs (UIMs). The two isoforms differ in their C-terminal parts (green), and DNAJB2b has a C-terminal geranylgeranyl moiety (GG) anchoring it to the endoplasmic reticulum. The extended DNAJB2a protein (p. * 278Glyext * 83) produced from the mutant allele has a C-terminal extension of 83 amino acids (magenta; sequence shown in the box). ( B ) RNA sequencing (RNAseq) coverage graphs covering the last exon(s) of the DNAJB2 transcripts. RNAseq of the proband (P) muscle sample showed equal expression of the wild-type and mutant alleles (arrow) and did not indicate splicing changes or altered isoform ratio compared with other samples run in the same batch (C1–5).

Techniques: Mutagenesis, Variant Assay, Ubiquitin Proteomics, Produced, Sequencing, RNA Sequencing, Expressing

$Western blotting of patient biopsy. ( A ) Western blotting of a muscle biopsy from the proband (P) revealed a reduced amount of both DNAJB2a and DNAJB2b proteins compared with pooled control (C), and no detectable mutant protein. P1 and P2 are independently prepared samples from the same biopsy. Post-blotting Coomassie staining of the myosin heavy chain (MYHC CBB) is shown as loading control. ( B ) Control (C1, C2) and proband (P) muscle biopsies were fractionated with the ProteoExtract Subcellular Proteome Extraction Kit to cytosolic (F1), membrane/organelle (F2), nuclear (F3) and cytoskeletal/insoluble (F4) fractions and analysed by western blotting. Total protein, tubulin, calnexin and histone 3 are shown as loading and fractionation controls. ( C ) The levels of DNAJB2 relative to total protein were quantified from the F1 and F2 fractions in (B) and represented normalized to the mean of control samples. Both DNAJB2 isoforms showed a ~50% reduction in the biopsy of the proband.$

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Western blotting of patient biopsy. ( A ) Western blotting of a muscle biopsy from the proband (P) revealed a reduced amount of both DNAJB2a and DNAJB2b proteins compared with pooled control (C), and no detectable mutant protein. P1 and P2 are independently prepared samples from the same biopsy. Post-blotting Coomassie staining of the myosin heavy chain (MYHC CBB) is shown as loading control. ( B ) Control (C1, C2) and proband (P) muscle biopsies were fractionated with the ProteoExtract Subcellular Proteome Extraction Kit to cytosolic (F1), membrane/organelle (F2), nuclear (F3) and cytoskeletal/insoluble (F4) fractions and analysed by western blotting. Total protein, tubulin, calnexin and histone 3 are shown as loading and fractionation controls. ( C ) The levels of DNAJB2 relative to total protein were quantified from the F1 and F2 fractions in (B) and represented normalized to the mean of control samples. Both DNAJB2 isoforms showed a ~50% reduction in the biopsy of the proband.

Techniques: Western Blot, Control, Mutagenesis, Staining, Extraction, Membrane, Fractionation

$Membrane localization of mutant DNAJB2. ( A ) In silico prediction. The amino acid sequence of DNAJB2a p. * 278Glyext * 83 was analysed with transmembrane helix prediction algorithms. The graph shows the scores from TMHMM (orange trace) and TMPRED (blue solid trace, in–out orientation, dashed trace, out–in) for the 83 amino acid extension. ( B ) Stably transfected C2C12 myotubes induced to express wild-type or p. * 278Glyext * 83 (ext) DNAJB2a, and non-induced control cells, were fractionated with the ProteoExtract Subcellular Proteome Extraction Kit to cytosolic (F1), membrane/organelle (F2), nuclear (F3) and cytoskeletal/insoluble (F4) fractions. Endogenous and overexpressed DNAJB2a were predominantly cytosolic, whereas p. * 278Glyext * 83 was enriched in the membrane fraction similarly to endogenous DNAJB2b. ( C ) T-REx 293 cells were transfected with a combination of wild-type DNAJB2a and DNAJB2b (a + b wt) or DNAJB2a p. * 278Glyext * 83 (ext) and fractionated. Wild-type DNAJB2a was mostly found in the cytosolic (CYT) fraction, whereas DNAJB2b and p. * 278Glyext * 83 were enriched in the microsomal (MIC) fraction, similarly to the ER marker calnexin. PNS, post-nuclear supernatant. ( D ) In transfected HeLa cells, wild-type (wt) V5-DNAJB2a showed diffuse nuclear and cytoplasmic localization, whereas p. * 278Glyext * 83 (ext) partially colocalized with the endoplasmic reticulum (ER) visualized with the Cytopainter ER staining kit.$

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Membrane localization of mutant DNAJB2. ( A ) In silico prediction. The amino acid sequence of DNAJB2a p. * 278Glyext * 83 was analysed with transmembrane helix prediction algorithms. The graph shows the scores from TMHMM (orange trace) and TMPRED (blue solid trace, in–out orientation, dashed trace, out–in) for the 83 amino acid extension. ( B ) Stably transfected C2C12 myotubes induced to express wild-type or p. * 278Glyext * 83 (ext) DNAJB2a, and non-induced control cells, were fractionated with the ProteoExtract Subcellular Proteome Extraction Kit to cytosolic (F1), membrane/organelle (F2), nuclear (F3) and cytoskeletal/insoluble (F4) fractions. Endogenous and overexpressed DNAJB2a were predominantly cytosolic, whereas p. * 278Glyext * 83 was enriched in the membrane fraction similarly to endogenous DNAJB2b. ( C ) T-REx 293 cells were transfected with a combination of wild-type DNAJB2a and DNAJB2b (a + b wt) or DNAJB2a p. * 278Glyext * 83 (ext) and fractionated. Wild-type DNAJB2a was mostly found in the cytosolic (CYT) fraction, whereas DNAJB2b and p. * 278Glyext * 83 were enriched in the microsomal (MIC) fraction, similarly to the ER marker calnexin. PNS, post-nuclear supernatant. ( D ) In transfected HeLa cells, wild-type (wt) V5-DNAJB2a showed diffuse nuclear and cytoplasmic localization, whereas p. * 278Glyext * 83 (ext) partially colocalized with the endoplasmic reticulum (ER) visualized with the Cytopainter ER staining kit.

Techniques: Membrane, Mutagenesis, In Silico, Sequencing, Stable Transfection, Transfection, Control, Extraction, Marker, Staining

Turnover studies. ( A – B ) Wild-type (wt) or p. * 278Glyext * 83 (ext) DNAJB2a were expressed in T-REx 293 cells alone or in combination, and their levels were assayed at 0, 2 and 4 h of cycloheximide treatment. (A) A representative experiment performed in triplicate. 100 and 50 indicate a normalization sample at 100% and 50% loading, common for all three blots. (B) Quantification of three replicate experiments. The level of DNAJB2 was normalized to the transfection marker (GFP-V5) and represented relative to the initial level ( t = 0). Each data point represents the mean ± SD of one experiment performed in triplicate. Asterisks indicate significant differences in remaining protein amount at t = 4 compared with wild-type (2-tailed t -test; * P = 0.029, * * P = 0.001). DNAJB2 p. * 278Glyext * 83 showed an increased turnover rate and also increased the turnover of the co-expressed wild-type DNAJB2a. ( C – D ) T-REx 293 cells expressing DNAJB2 p. * 278Glyext * 83 were treated with cycloheximide alone (CH) or in combination with the proteasome inhibitor MG132 (MG) or lysosomal inhibitors (NH 4 Cl/leupeptin; NL) for 2 h. (D) The level of DNAJB2 was normalized to the transfection marker (GFP-V5) and represented relative to the initial level ( t = 0). The graph shows means ± SD from three replicate experiments, each performed in triplicate. MG132 efficiently blocked the turnover of mutant DNAJB2 ( * * P = 0.006, 2-tailed t -test).

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Turnover studies. ( A – B ) Wild-type (wt) or p. * 278Glyext * 83 (ext) DNAJB2a were expressed in T-REx 293 cells alone or in combination, and their levels were assayed at 0, 2 and 4 h of cycloheximide treatment. (A) A representative experiment performed in triplicate. 100 and 50 indicate a normalization sample at 100% and 50% loading, common for all three blots. (B) Quantification of three replicate experiments. The level of DNAJB2 was normalized to the transfection marker (GFP-V5) and represented relative to the initial level ( t = 0). Each data point represents the mean ± SD of one experiment performed in triplicate. Asterisks indicate significant differences in remaining protein amount at t = 4 compared with wild-type (2-tailed t -test; * P = 0.029, * * P = 0.001). DNAJB2 p. * 278Glyext * 83 showed an increased turnover rate and also increased the turnover of the co-expressed wild-type DNAJB2a. ( C – D ) T-REx 293 cells expressing DNAJB2 p. * 278Glyext * 83 were treated with cycloheximide alone (CH) or in combination with the proteasome inhibitor MG132 (MG) or lysosomal inhibitors (NH 4 Cl/leupeptin; NL) for 2 h. (D) The level of DNAJB2 was normalized to the transfection marker (GFP-V5) and represented relative to the initial level ( t = 0). The graph shows means ± SD from three replicate experiments, each performed in triplicate. MG132 efficiently blocked the turnover of mutant DNAJB2 ( * * P = 0.006, 2-tailed t -test).

Techniques: Transfection, Marker, Expressing, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Oligomerization of DNAJB2. ( A ) Local sequence alignment of DNAJB2a (B2a) and DNAJB6b (B6b), with the coloured shadings indicating the J domain (JD), the glycine/phenylalanine-rich domain (G/F), the serine-rich region (SR) and the C-terminal domain (CTD) as defined for DNAJB6b by Karamanos et al . . Most of the region mediating DNAJB6b oligomerization (CTD β strands β1–β5) is highly similar between the two proteins. The C-terminal part of DNAJB2a (amino acids 218–277), not homologous to DNAJB6, is not shown. ( B – C ) Density gradient centrifugation of T-REx 293 cell lysates in 10–80% sucrose gradients. Fractionation profile of wild-type V5-DNAJB2a (a wt) suggests its oligomerization into polydisperse oligomers, similarly to endogenous DNAJB6 (a and b isoforms indicated) in the same samples (B). Co-expressed untagged wild-type (wt) and p. * 278Glyext * 83 (ext) DNAJB2a are both distributed throughout the gradient, albeit with somewhat different profiles (C). T, total samples.

Techniques: Sequencing, Gradient Centrifugation, Fractionation

Review

Structured Review

Images

1) Product Images from "Impaired DNAJB2 Response to Heat Shock in Fibroblasts from a Neuropathy Patient with DNAJB2/HSJ1 Mutation: Cystamine as a Potential Therapeutic Intervention"

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