Anti-PROM1 Antibody Picoband®

Western blot analysis of PROM1 using anti-PROM1 antibody (PB9156).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human CACO-2 whole cell lysates,
Lane 2: human SW620 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-PROM1 antigen affinity purified polyclonal antibody (Catalog # PB9156) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for PROM1 at approximately 120KD. The expected band size for PROM1 is at 120KD.

MCF-7 ALDH1A1 affects in vitro stemness. a Representative images of tumorspheres (4x magnification) showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. b Representative images of tumorspheres (4x magnification) of MCF-7 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + , showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. b1, b2, b3. Representative images of tumorspheres (10x magnification) of MCF-1 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + , showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. c Quantification of MCF-7 tumorspheres. Tumorspheres area were calculated using ImageJ Software. Ten pictures for each well were quantified. Tumorspheres> 10.000 pixel square were considered. ** p < 0.01 vs MCF-7 Scr. ### p < 0.001 vs MCF-7 ALDH1A1KD. ( n = 3). d Western blot analysis of stemness markers CD133 and KLF4 in MCF-7 Scr, MCF-7 ALDH1A1KD, and MCF-7 ALDH1A1 + tumorspheres. e Western blot analysis of ALDH1A1, HIF-1α and VEGF in MCF-7 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + tumorspheres
Index in PubMed under a CC BY license. PMID: 30541574

MCF-7 ALDH1A1 regulates angiogenic factor output via retinoic acid signalling. a Angiogenic factor release evaluated by ELISA plate array in supernatants of MCF-7 treated with CM037 (1 μM) for 48 h. The experiment was performed 2 times in duplicate. b MCF-7 cells were exposed to CM037 at different concentrations (1 and 10 μM) for 18 h and western blot was carried out. β-Actin was used to normalize loading. c Cells were treated with CM037 (1 μM, 18 h) and VEGF levels were measured by ELISA assay in MCF-7 conditioned media. After 18 h supernatants were harvested and cells fixed, stained and counted. The number of counted cells was not significantly different. Data are reported as pg/ml. ** p < 0.01 vs untreated cells. d RT-PCR analysis of VEGF in MCF-7 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + cultured in medium with 1% FBS for 48 h. Data are reported as ΔCt (Ct gene of interest-Ct Housekeeping gene). *** p < 0.001 vs MCF-7 Scr. ### p < 0.001 vs MCF-7 ALDH1A1KD. e Western blot analysis of VEGF and HIF-1α in MCF-7 exposed or not to CoCl 2 (100 μM, 72 h, 1% FBS). β-Actin was used as loading control. Gel shown is representative of three experiments with similar results. f Quantification of blots reported in e . * p < 0.05 vs MCF-7 Scr. ** p < 0.01 vs MCF-7 Scr. ### p < 0.001 vs MCF-7 ALDH1A1KD. g Soluble VEGF was detected by ELISA in media conditioned by MCF-7 cells. Cells were seeded in 24-well plates at density 3 × 10 4 cells/well. After 48 h the supernatants were harvested and cells fixed, stained and counted. The number of counted cells was not significantly different. Data are reported as pg/ml. ** p < 0.01 vs MCF-7 Scr. ## p < 0.01 vs MCF-7 ALDH1A1KD. h HIF-1α and VEGF expression evaluated by western blot in MCF-7 ALDH1A1KD cells exposed for 48 h (1 μM) to exogenous retinoic acid. i HIF-1α and VEGF expression in MCF-7 ALDH1A1 + treated with RAR antagonist (AGN193109) and RXR antagonist (UVI 3003) for 48 h (each at 1 μM). β-Actin was used as loading control. Gel shown is representative of three experiments with similar results. j VEGF and CD133 expression in MCF-7 transiently silenced for HIF-1α. β-Actin was used as loading control. Gel shown is representative of three experiments with similar results
Index in PubMed under a CC BY license. PMID: 30541574

ALDH1A1 influences tumor angiogenesis and VEGF production in vivo. a Evaluation of VEGF, HIF-1α and ALDH1A1 RNA in tumor samples. Frozen tumors were homogenized and RNA was extracted to perform RT-PCR analysis of VEGF, HIF-1α and ALDH1A1 mRNA. Data are reported as ΔCt (Ct gene of interest-Ct Housekeeping gene). Each bar is the mean of 6 different tumors. The experiment was repeated two times. * p < 0.05 vs Scr group. ** p < 0.01 vs Scr group. # p < 0.05 vs ALDH1A1KD group. ### p < 0.001 vs ALDH1A1KD group. b Evaluation of VEGF and ALDH1A1 proteins in tumor samples. Tissues were harvested, homogenized and sonicated. Subsequently, proteins were extracted and western blot was performed. β-Actin was used as loading control. The experiment was repeated two times. c Evaluation of mRNA for CAIX (HIF-1α target gene) and stemness markers (SOX2, NANOG, OCT-4 and TWIST) in tumor samples. Each bar is the mean of 6 different tumors. The experiment was repeated two times. # p < 0.05 vs ALDH1A1KD group. ## p < 0.01 vs ALDH1A1KD group. ### p < 0.001 vs ALDH1A1KD group. d Evaluation of HIF-1α and stemness markers (CD133, KLF4 and SOX2) proteins in tumor samples. The experiment was repeated two times. e Quantification of blots reported in d . * p < 0.05 vs Scr group. # p < 0.05 vs ALDH1A1KD group. ## p < 0.01 vs ALDH1A1KD group. f Quantification of microvessel density by human CD31 staining (magnification 20x) was done counting 5 random fields for section, each slide having five sections. ** p < 0.01 vs Scr group. ## p < 0.01 vs ALDH1A1 + group. g Representative images of immunostaining for CD31 (red) and DAPI (blue) in tumor sections from Scr (left), ALDH1A1KD (center) or ALDH1A1 + (right) mice. Pictures report different vessel densities in tumors. Magnification 20x. Scale bar, 50 μm. h Representative images of double-immunostaining for CD31 (red) and NG2 (green) in tumor sections from Scr (left), ALDH1A1KD (center) or ALDH1A1 + (right) mice. DAPI staining is blue. Magnification 40x. Scale bars, 50 μm
Index in PubMed under a CC BY license. PMID: 30541574

IHC analysis of PROM1 using anti-PROM1 antibody (PB9156).
PROM1 was detected in paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-PROM1 Antibody (PB9156) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of PROM1 using anti-PROM1 antibody (PB9156).
PROM1 was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-PROM1 Antibody (PB9156) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

Flow Cytometry analysis of CACO-2 cells using anti-PROM1 antibody (PB9156).
Overlay histogram showing CACO-2 cells stained with PB9156 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-PROM1 Antibody (PB9156,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.