Journal: Cellular and Molecular Life Sciences

Article Title: The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

doi: 10.1007/s00018-010-0452-1

Figure Lengend Snippet: Compound 11 inhibits Hsp27 phosphorylation by MK3 and MK5, but not by MK2. Recombinant Hsp27 was incubated with purified MK2, MK3, or MK5 in the presence or absence of compound 11 . Hsp27 phosphorylation at serine residue 78 (p-Ser78 Hsp27) was monitored by western blot with phosphoserine-78 specific antibodies. Lanes 1 , 3 , and 5 : Hsp27 incubated with purified MK5, MK3, and MK2, respectively. Lanes 2 , 4 , and 6 : as lanes 1 , 3 , and 5 but in the presence of 20 μM noroxoaconitine (compound 11 )

Article Snippet: Recombinant Hsp27 was purchased from Signal Chem (Richmond, BC, Canada).

Techniques: Phospho-proteomics, Recombinant, Incubation, Purification, Residue, Western Blot

Journal: Nature cell biology

Article Title: Small heat shock proteins operate as molecular chaperones in the mitochondrial intermembrane space.

doi: 10.1038/s41556-022-01074-9

Figure Lengend Snippet: Fig. 5 | The mitochondrial interactome of HSPB1 is enriched for transmembrane proteins of the inner mitochondrial membrane. a, Volcano plot of the interactome of HSPB1S135F versus eGFP obtained from WCL analysis. Mitochondrial proteins are highlighted in red. A one-sided t-test was performed for pairwise comparison of both conditions. Significantly enriched proteins are indicated by curved line, set by an FDR value of 0.05 and S0 value of 1. b, Co-immunoprecipitation with anti-V5 beads to validate the mitochondrial interactors in WCL. c, Co-immunoprecipitation with anti-V5 beads to validate the mitochondrial interactors in mitochondria that were pre-treated with proteinase K (10 μg ml−1) to remove all non-imported proteins. d, Co-immunoprecipitation with anti-V5 beads to validate the interaction between V5-tagged HSPB1 wild-type (WT), HSPB1 mutants R127W (RW), S135F (SF) or P182L (PL) and SLC25A12. GFP-V5 was used as negative control. e, Determination of the binding sites of HSPB1

Article Snippet: Untagged sHSPs HSPB1 to HSPB10 (Frt-HSPBx, Addgene plasmids #63092–#63101, Addgene) were a kind gift from Harm Kampinga (University of Groningen).

Techniques: Membrane, Comparison, Immunoprecipitation, Negative Control, Binding Assay

Journal: PloS one

Article Title: Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

doi: 10.1371/journal.pone.0066001

Figure Lengend Snippet: Figure 1. Characterization of hHSP27. A–B, Isolated human heat shock protein 27 (hHSP27) or recombinant HSP27 (rHSP27) proteins (1 mg or 10 mg) were separated by SDS-PAGE (A) and native-PAGE (B) and stained with Coomassie brilliant blue. C, hHSP27 proteins were immunoblotted with antibodies against HSP27, phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27. D, hHSP27 proteins were separated by native-PAGE and immunoblotted with antibodies against ab-crystallin and HSP20. E, rHSP27 (10 ng), hHSP27 (10 ng), ab-crystallin (5 ng), and HSP20 (5 ng) were separated by SDS-PAGE followed by immunoblotting with antibodies against HSP27, ab-crystallin, and HSP20. doi:10.1371/journal.pone.0066001.g001

Article Snippet: The observers of the behavioral tests were blinded to the treatment groups, and mice of the various groups were randomized during a given testing period. hHSP27 and HSP27 Antibody, HSP27 Elution Peptide Administration or Recombinant HSP27 Administration Mice received intravenous injections of 50 mg of hHSP27 mixed with 50 or 500 mg of HSP27-N1 or -C1 antibody 1 h after reperfusion (n= 3 in each group), 5 or 50 mg of HSP27-N1 and - C1 peptides, which were used in the elution, intravenously 1 h after reperfusion (n= 3 in each group), or 50 mg of recombinant HSP27 (rHSP27; Acris Antibodies GmbH) 1 h after reperfusion (n= 3).

Techniques: Isolation, Recombinant, SDS Page, Clear Native PAGE, Staining, Western Blot

Journal: PloS one

Article Title: Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

doi: 10.1371/journal.pone.0066001

Figure Lengend Snippet: Figure 4. Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects. A, Infarct volumes in control, hHSP27 (50 mg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means6SEM of 3 mice in each group. **P,0.001 vs. controls. B– D, Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDS- PAGE (B) and native-PAGE (C), stained with Coomassie brilliant blue (B,C), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (D). E, Photomicrographs of infarct areas stained with cresyl violet in hHSP27 and dephosphorylated hHSP27 groups prepared 24 h after reperfusion. Scale bar = 1 mm. hHSP27, human heat shock protein. doi:10.1371/journal.pone.0066001.g004

Article Snippet: The observers of the behavioral tests were blinded to the treatment groups, and mice of the various groups were randomized during a given testing period. hHSP27 and HSP27 Antibody, HSP27 Elution Peptide Administration or Recombinant HSP27 Administration Mice received intravenous injections of 50 mg of hHSP27 mixed with 50 or 500 mg of HSP27-N1 or -C1 antibody 1 h after reperfusion (n= 3 in each group), 5 or 50 mg of HSP27-N1 and - C1 peptides, which were used in the elution, intravenously 1 h after reperfusion (n= 3 in each group), or 50 mg of recombinant HSP27 (rHSP27; Acris Antibodies GmbH) 1 h after reperfusion (n= 3).

Techniques: De-Phosphorylation Assay, Control, Recombinant, SDS Page, Clear Native PAGE, Staining

Journal: bioRxiv

Article Title: Identification of novel human cellular substrates of Staphylococcus aureus serine protease SplB

doi: 10.1101/2025.09.09.675177

Figure Lengend Snippet: (A) Silver-stained gel for serpin A12 and sodium/potassium-transporting ATPase subunit α-2 (EEFA1) incubated with SplB WT or SplB Ser195Ala (mut) at equimolar for 24 hours (B) Silver-stained gel of the In vitro cleavage products after incubating 1.5 µM substrate candidates with different concentrations of SplB WT at various time points. Incubation with SplB Ser195Ala served as control. Substrate candidates included desmin (∼55 kDa), recombinant IGKC (constant domain of the immunoglobulin κ light chain (∼12 kDa) fused to a glutathione S-transferase tag (GST; ∼26 kDa), α-enolase (ENOA∼ 49.3 kDa), and heat shock protein β1 (HSPB1∼ 28 kDa). The experiment was performed with n = 4 technical replicates, and a typical example is shown. Conditions included: 0: Substrate only (No SplB!); 1: SplB WT and substrate at equimolar concentrations, 4-hour incubation; 2: SplB WT and substrate at equimolar concentrations, 24-hour incubation; 3: SplB WT 4:1 substrate-to-enzyme molar ratio, 24-hour incubation; 4: SplB Ser195Ala and substrate at equimolar concentrations, 24-hour incubation. *= cleavage product != full length protein substrate #= SplB. Gel images were cropped for clarity, indicated by blue dotted lines; full gels are available upon request.

Article Snippet: All substrate candidates were commercially available and purchased in recombinantly purified form: desmin (Progen, Germany), heat shock protein β1 (Creative Biomart, USA), α-enolase (Creative Biomart, USA), the constant domain of the immunoglobulin κ-chain (Antibodies-online.com, Germany), serpin A12 (Creative Biomart, USA), and human IgG isotype (Invitrogen, Germany) along with two additional proteins from the same family as desmin: vimentin (Thermo Fisher Scientific, USA) and nestin (Rockville, USA).

Techniques: Staining, Incubation, In Vitro, Control, Recombinant

Journal: bioRxiv

Article Title: Identification of novel human cellular substrates of Staphylococcus aureus serine protease SplB

doi: 10.1101/2025.09.09.675177

Figure Lengend Snippet: In vitro cleavage products after incubating 1.5 µM of whole IgG isotype solution with different concentrations of SplB- WT at various time points, alongside SplB Ser195Ala . Reduction with β-mercaptoethanol resulted in the separation of IgG into its constituent chains. Two major bands are visible: the heavy chain at approximately 50 kDa and the light chain at approximately 25 kDa. The experiment was repeated with n = 2 technical replicates. Conditions included: 0: Substrate Only (No SplB); 1: SplB WT and substrate at equimolar concentrations, 4-hour incubation; 2: SplB WT and substrate at equimolar concentrations, 24-hour incubation; 3: SplB WT 4:1 substrate-to-enzyme molar ratio, 24-hour incubation; 4: SplB Ser195Ala and substrate at equimolar concentrations, 24-hour incubation. *= cleavage product, != IgG heavy chain, !!= IgG light chain, #= SplB

Article Snippet: All substrate candidates were commercially available and purchased in recombinantly purified form: desmin (Progen, Germany), heat shock protein β1 (Creative Biomart, USA), α-enolase (Creative Biomart, USA), the constant domain of the immunoglobulin κ-chain (Antibodies-online.com, Germany), serpin A12 (Creative Biomart, USA), and human IgG isotype (Invitrogen, Germany) along with two additional proteins from the same family as desmin: vimentin (Thermo Fisher Scientific, USA) and nestin (Rockville, USA).

Techniques: In Vitro, Incubation

Journal: bioRxiv

Article Title: Identification of novel human cellular substrates of Staphylococcus aureus serine protease SplB

doi: 10.1101/2025.09.09.675177

Figure Lengend Snippet: In vitro cleavage products after incubating 1.5 µM of vimentin (∼54 kDa) and nestin (>170 kDa) with different concentrations of SplB WT at various time points. SplB Ser195Ala served as control. The experiment was performed with n = 3 technical replicates, a typical gel is shown. Conditions included: 0: No SplB; 1: SplB WT and substrate at equimolar concentrations, 4-hour incubation; 2: SplB WT and substrate at equimolar concentrations, 24-hour incubation; 3: SplB WT 4:1 substrate-to-enzyme molar ratio, 24-hour incubation; 4: SplB Ser195Ala and substrate at equimolar concentrations, 24-hour incubation. *= cleavage product != full length protein substrate #= SplB.

Article Snippet: All substrate candidates were commercially available and purchased in recombinantly purified form: desmin (Progen, Germany), heat shock protein β1 (Creative Biomart, USA), α-enolase (Creative Biomart, USA), the constant domain of the immunoglobulin κ-chain (Antibodies-online.com, Germany), serpin A12 (Creative Biomart, USA), and human IgG isotype (Invitrogen, Germany) along with two additional proteins from the same family as desmin: vimentin (Thermo Fisher Scientific, USA) and nestin (Rockville, USA).

Techniques: In Vitro, Control, Incubation

Journal: bioRxiv

Article Title: Identification of novel human cellular substrates of Staphylococcus aureus serine protease SplB

doi: 10.1101/2025.09.09.675177

Figure Lengend Snippet: Cell lysates of human myotubes were prepared and the ability of 47.2 ng of SplB WT to cleave 100 ng of native and recombinantly expressed desmin was assessed after 24 h of incubation. Conditions included: 0: Lysate or substrate only (No SplB); 1: SplB WT and substrate at equimolar concentrations, 24-hour incubation; 2: SplB Ser195Ala and substrate at equimolar concentrations, 24-hour incubation. A Western blot was performed using primary polyclonal anti-desmin antibodies (rabbit), detected with secondary goat anti-rabbit RD680 antibody (red color). A fluorescently labeled TactinXT CW800) (green color) was used to visualize the Twin-Strep tagged SplB proteins.

Article Snippet: All substrate candidates were commercially available and purchased in recombinantly purified form: desmin (Progen, Germany), heat shock protein β1 (Creative Biomart, USA), α-enolase (Creative Biomart, USA), the constant domain of the immunoglobulin κ-chain (Antibodies-online.com, Germany), serpin A12 (Creative Biomart, USA), and human IgG isotype (Invitrogen, Germany) along with two additional proteins from the same family as desmin: vimentin (Thermo Fisher Scientific, USA) and nestin (Rockville, USA).

Techniques: Incubation, Western Blot, Labeling

Journal: bioRxiv

Article Title: Identification of novel human cellular substrates of Staphylococcus aureus serine protease SplB

doi: 10.1101/2025.09.09.675177

Figure Lengend Snippet: HUNTER analysis was used to identify SplB cleavage sites by comparing N-terminal peptide enrichment across conditions. The x-axis shows peptide enrichment in SplB WT –treated versus untreated substrate pools, and the y-axis shows enrichment in SplB WT versus SplB Ser195Ala (≥2-fold). Data represents three biological replicates. Red dots mark peptides ≥2-fold enriched in both comparisons, indicating specific SplB WT cleavage sites. Identified sites included desmin (Q133), heat shock protein β1 (Q176), ( , , ) nestin (Q33, Q1560, Q1491), and vimentin (Q82). Corresponding peptide sequences were aligned to the protein sequence; the P1 residue (N-terminal of the peptide) was deduced and found to be Q or E. Sequences C-terminal of the cleavage sites, containing residues at P1′, P2′, P3′, etc., are shown.

Article Snippet: All substrate candidates were commercially available and purchased in recombinantly purified form: desmin (Progen, Germany), heat shock protein β1 (Creative Biomart, USA), α-enolase (Creative Biomart, USA), the constant domain of the immunoglobulin κ-chain (Antibodies-online.com, Germany), serpin A12 (Creative Biomart, USA), and human IgG isotype (Invitrogen, Germany) along with two additional proteins from the same family as desmin: vimentin (Thermo Fisher Scientific, USA) and nestin (Rockville, USA).

Techniques: Sequencing, Residue

Journal: Scientific Reports

Article Title: Heat Shock Protein 27 is down-regulated in Ballooned Hepatocytes of Patients with Nonalcoholic Steatohepatitis (NASH)

doi: 10.1038/srep22528

Figure Lengend Snippet: Bars represent immunoreactivity scores of HSP27 staining; mean ± SD values. ( A ) Representative liver expression pattern of HSP27 in a NAFLD patient without ballooning degeneration; black arrows indicate HSP27 immunoreactivity in hepatocyte cytoplasm or surrounding the lipid droplets showing a granular pattern. ( B ) Representative liver expression pattern of HSP27 in a patient with NASH and ballooning degeneration. ( C ) Representative liver expression pattern of HSP27 in focal areas of marked ballooning degeneration (C1) or absence of ballooning (C2) in the same liver specimen of a patients with NAFLD. BH: ballooned hepatocytes (enlarged hepatocytes with pale to clear cytoplasm, some of them showing glycogenated nuclei GN); LD: lipid droplet. Protein expression was explored using immunohistochemistry; ballooning degeneration was dichotomized to: ballooning 0 (none) versus ballooning 1 (mild or marked). HSP27 immunoreactivity was examined using light microscopy of liver sections; counterstaining was performed with hematoxylin. Original magnification: 400X.

Article Snippet: Following microwave heat-induced epitope retrieval in 0.1 M citrate buffer at pH 6.0 for 20 min, the slides were incubated with a dilution of 1:100 of HSP27 antibody-C-terminal region ARP30177_T100 (Aviva Systems Biology, San Diego, CA, USA).

Techniques: Staining, Expressing, Immunohistochemistry, Light Microscopy

Journal: Scientific Reports

Article Title: Heat Shock Protein 27 is down-regulated in Ballooned Hepatocytes of Patients with Nonalcoholic Steatohepatitis (NASH)

doi: 10.1038/srep22528

Figure Lengend Snippet: Primer sequences used for mRNA expression analysis.

Article Snippet: Following microwave heat-induced epitope retrieval in 0.1 M citrate buffer at pH 6.0 for 20 min, the slides were incubated with a dilution of 1:100 of HSP27 antibody-C-terminal region ARP30177_T100 (Aviva Systems Biology, San Diego, CA, USA).

Techniques: Expressing