anti hspb1  (Proteintech)

Journal: Frontiers in Molecular Biosciences

Article Title: ZKSCAN3 promotes ovarian cancer cell proliferation by increasing HSPB1 expression

doi: 10.3389/fmolb.2025.1623062

Figure Lengend Snippet: HSPB1 expression is regulated by ZKSCAN3. (A) Shown is a snapshot of the UCSC genome browser that illustrates the ChIP-seq signals from Input and ZKSCAN3-ChIP at the HSPB1 locus. DNase signal was from ENCODE. (B) Shown is a snapshot of Integrative Genomics Viewer, which illustrates the RNA-Seq signals at the HSPB1 locus. (C) The HSPB1 mRNA levels in the control and ZKSCAN3-KD cells were analyzed with real-time RT-PCR. ZKSCAN3 mRNA was included to show knockdown efficiency. mRNA levels presented were normalized to ACTB (β-actin). (D) Cells were analyzed with ChIP. Immunoprecipitation was done with the ZKSCAN3 antibody with IgG as the control. The left panel shows the result of real-time PCR, and the right panel shows the agarose gel image for conventional PCR products. (E) Control or ZKSCAN3-KD cells were analyzed with ChIP followed by real-time PCR. Immunoprecipitation was done with the ZKSCAN3 antibody. IgG was included as a control. (F) ZKSCAN3 was knocked down with lentivirus-expressed shRNA. The whole-cell lysate (WCL) was then analyzed by Western blot (WB). (G) ZKSCAN3 was stably expressed in HEY ovarian cancer cells with lentivirus transduction followed by antibiotic selection. The whole-cell lysate (WCL) was then analyzed by Western blot (WB). (H) Shown is linear regression for protein levels of ZKSCAN3 and HSPB1 in 85 ovarian cancer patients from the CPTAC (PDC000110).

Article Snippet: After blocking with 1% BSA, samples were incubated with primary antibodies against Myc tag (Proteintech 16286-1-AP) or HSPB1 (Proteintech #18284-1-AP).

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, Control, Quantitative RT-PCR, Knockdown, Immunoprecipitation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, shRNA, Western Blot, Stable Transfection, Transduction, Selection

Journal: Frontiers in Molecular Biosciences

Article Title: ZKSCAN3 promotes ovarian cancer cell proliferation by increasing HSPB1 expression

doi: 10.3389/fmolb.2025.1623062

Figure Lengend Snippet: HSPB1 contributes to ZKSCAN3-mediated promotion of cell proliferation. (A) Survival analysis for 84 patients in the CPTAC ovarian cancer cohort. Patients were divided into “HSPB1 high” and “HSPB1 low” groups based on HSPB1 protein levels as determined by mass spectrometry. Median HSPB1 level was used as a threshold. (B) Cells were fractionated into cytoplasm (c), nuclear extract (NE), and nuclear pellet (NP) fractions. Equal amounts of these fractions were analyzed with WB. GAPDH and histone H3 were included as markers for cytoplasm and nuclei, respectively. (C) Control and HSPB1-KD cells were subject to immunofluorescence analysis with the HSPB1 antibody. Nuclei were counterstained with DAPI. Scale bars denote 10 μm. (D) Myc-tag HSPB1 was stably expressed in HEY cells with lentivirus transduction followed by antibiotic selection. Cells were then subject to immunofluorescence analysis with Myc-tag antibody. Nuclei were counterstained with DAPI. Scale bars denote 10 μm. (E) HSPB1 was knocked down in ovarian cancer cells with lentivirus-expressed shRNA. Cell proliferation over 4 days was measured with cell counting. (F) HSPB1 was knocked down in ovarian cancer cells with lentivirus-expressed shRNA. Each 3.5-cm dish was seeded with 1,000 cells. Cells were fixed with crystal violet 12 days later. (G) Control or HSPB1-knockdown cells were labeled with BrdU and then subject to immunostaining with anti-BrdU antibody. The left panel shows representative images, while the right panel shows the statistical results for four independent images. Scale bars denote 10 μm. (H) HSPB1 was overexpressed in ovarian cancer cells. Cell proliferation over 5 days was measured with cell counting. (I) HSPB1 was overexpressed in ovarian cancer cells. Each 3.5-cm dish was seeded with 1,000 cells. Cells were fixed with crystal violet 12 days later. (J) HSPB1 was expressed with lentivirus transduction in ZKSCAN3-KD cells. Cell proliferation over 4 days was then measured with cell counting. (K) HSPB1 was rescue expressed with lentivirus in ZKSCAN3-KD cells. Each 3.5-cm dish was seeded with 1,000 cells. Cells were fixed with crystal violet 12 days later.

Article Snippet: After blocking with 1% BSA, samples were incubated with primary antibodies against Myc tag (Proteintech 16286-1-AP) or HSPB1 (Proteintech #18284-1-AP).

Techniques: Mass Spectrometry, Control, Immunofluorescence, Stable Transfection, Transduction, Selection, shRNA, Cell Counting, Knockdown, Labeling, Immunostaining