skmel28  (ATCC)

Journal: Antibodies

Article Title: A Recombinant Antibody Against Human DRP1 Serine 616 Phosphorylation Enables Detection of BRAF V600E -Associated Mitochondrial Division in Cancer

doi: 10.3390/antib15020038

Figure Lengend Snippet: Hybridoma screenings and recombinant 3G11 antibody evaluation. ( A ) Hybridomas generated from the top five anti-sera were evaluated for specificity using Western blot analysis of full-length recombinant GST-DRP1 ± ERK1 treatment. 100 ng of recombinant GST-DRP1 was loaded per lane. Total DRP1 was evaluated for equal protein loading. ( B , C ) The hybridomas were screened using Western blot analysis of whole cell lysates (100 µg/lane; ( B )) or heavy membrane fractions (25 µg/lane; ( C )) isolated from SKMEL28 and A375 cells treated with GSK1120212 (50 nM; GSK), PLX4032 (1 µM; PLX), or DMSO for 6 h. Total DRP1 was evaluated for equal protein loading. ( D ) Primary melanoma lines were treated with GSK1120212 (50 nM) or PLX4032 (1 µM) for 6 h. Whole cell lysates were Western blotted for the indicated proteins. Recombinant 3G11 was evaluated for DRP1-S616Ⓟ detection. Actin was probed for equal protein loading. ( E ) A375 and SKMEL28 were treated with GSK1120212 (50 nM) or PLX4032 (1 µM) for 6 h and evaluated as in D . Recombinant 3G11 was evaluated for DRP1-S616Ⓟ detection. Actin was probed for equal protein loading. ( F ) Heavy membrane fractions from the same treatments in D were Western blotted for the indicated proteins. Recombinant 3G11 was evaluated for DRP1-S616Ⓟ. HSP60 was probed for equal protein loading. Molecular weight standards for all Western blots are indicated in kilodaltons (kDa). ( G , H ) YUPEET cells expressing two independent shRNAs targeting DNM1L (sh1, sh2), or their combination, were analyzed by RT-qPCR and Western blot to determine DNM1L knockdown and loss of detection by recombinant 3G11. ( I ) YUPEET cells were treated with GSK1120212 (50 nM) or DMSO for 6 h, and whole cell lysates were evaluated by SDS-PAGE and Western blot for the indicated proteins to assess the specificity of recombinant 3G11 for DRP1-S616 phosphorylation relative to S637. Actin was used as a loading control. ( J ) YUPEET cell lysates were treated with λ-phosphatase for 30 min prior to SDS-PAGE and Western blot analysis. Actin was used as a loading control. ( K ) Untreated whole cell lysates (25 and 50 μg) from YUPEET cells were analyzed by SDS-PAGE and Western blot to compare the sensitivity of recombinant 3G11 and the Cell Signaling Technology (CST) antibody. Total DRP1 and actin were used as loading controls. Data are representative of three independent experiments.

Article Snippet: A375 and SKMEL28 human-derived non-primary (i.e., lymph node and secondary metastasis-derived, respectively) melanoma lines were purchased from ATCC and cultured in DMEM media.

Techniques: Recombinant, Generated, Western Blot, Membrane, Isolation, Molecular Weight, Expressing, Quantitative RT-PCR, Knockdown, SDS Page, Phospho-proteomics, Control

Journal: Antibodies

Article Title: A Recombinant Antibody Against Human DRP1 Serine 616 Phosphorylation Enables Detection of BRAF V600E -Associated Mitochondrial Division in Cancer

doi: 10.3390/antib15020038

Figure Lengend Snippet: Oncogenic MAPK-regulated DRP1-S616Ⓟ expression is detected by recombinant 3G11. ( A ) SKMEL28 cells were treated with GSK1120212 (50 nM) for 16 h, fixed, and stained for TOMM20 (green; mitochondria) and Hoechst 33342 (blue; nuclei). Scale bars = 10 microns. ( B ) SKMEL28 cells were studied as in ( A ). Mitochondrial networks are shown as maximum intensity projections of super-resolution Leica LIGHTNING deconvolved confocal z-stacks. Imaris surface features were used to generate 3D projections to visualize changes in mitochondrial network architecture. Scales bars = 5 microns. ( C ) Mitochondrial elongation was quantified using the Perimeter: Area Ratio. ( D , E ) SKMEL28 cells were treated with GSK1120212 (50 nM) for 16 h, fixed, and stained for TOMM20 (green), DRP1-S616Ⓟ (red), and Hoechst 33342 (blue). Red channel intensity was quantified to assess DRP1-S616Ⓟ signal intensity using Mander’s coefficient. Scale bars = 10 microns. Zoomed network images (white dashed box) = 50 micron scale bars. ( F , G ) A375 cells were analyzed as panels ( D , E ). All experiments were performed at least twice, with statistical significance determined from a minimum of two independent experiments using a one-way ANOVA test. ** refers to a p value ≤ 0.01 and **** refers to a p value ≤ 0.0001.

Article Snippet: A375 and SKMEL28 human-derived non-primary (i.e., lymph node and secondary metastasis-derived, respectively) melanoma lines were purchased from ATCC and cultured in DMEM media.

Techniques: Expressing, Recombinant, Staining