ppp3ca polyclonal antibody  (Elabscience Biotechnology)

Journal: HemaSphere

Article Title: A microRNA expression signature in infant t(4;11) KMT2A::AFF1+ BCP‐ALL uncovers novel therapeutic targets

doi: 10.1002/hem3.70353

Figure Lengend Snippet: MiR‐194, miR‐99b, and miR‐125a‐5p act through their downstream targets CA5B, PPP3CA, and PPP2R5C. (A) CA5B, PPP3CA, and PPP2R5C are predicted targets of miR‐194, miR‐99b, and miR‐125a‐5p, respectively. (B) CA5B , (C) PPP3CA, and (D) PPP2R5C expression in leukemic blasts, non‐blasts from KMT2A::AFF1+ B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) patients, and normal FL pro‐B lymphoid cells. Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) of (E) CA5B , (F) PPP3CA, and (G) PPP2R5C in SEM cells that overexpress miRVANA® mimics ( n ≥ 3). (H) Western blots of SEM cells transfected with miRVANA® mimics for CA5B, PPP3CA, and PPP2R5C. The relative quantification (RQ) according to the control condition is presented. β‐Actin was used as a reference gene. (I) Luciferase assay in 293T cells using pGL3‐promoter‐UTR vectors of CA5B, PPP3CA, and PPP2R5C to assess microRNA‐mediated regulation ( n ≥ 3). (J) Proliferation and (K) apoptosis of SEM CRISPR‐Cas9 cells knocked out with CA5B, PPP3CA, and PPP2R5C ( n ≥ 3). Data are presented as mean ± SEM and compared using a Mann–Whitney U test with a bilateral P‐value according to the indicated reference value (REF): P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Article Snippet: The primary antibody was added at a dilution of 1:1000 in 5% milk TBST Buffer and incubated overnight at 4°C, with mild shaking: human Carbonic Anhydrase VB/CA5B antibody (Biotechne Cat# AF3176), rabbit anti‐PPP2R5C antibody affinity purified (Bethyl Laboratories Inc Cat# A303‐814A), PPP3CA polyclonal antibody (Elabscience Cat# E‐AB‐14813), and monoclonal anti‐β‐actin antibody produced in mouse (Sigma Cat# A2228).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Quantitative Proteomics, Control, Luciferase, CRISPR, MANN-WHITNEY

Journal: HemaSphere

Article Title: A microRNA expression signature in infant t(4;11) KMT2A::AFF1+ BCP‐ALL uncovers novel therapeutic targets

doi: 10.1002/hem3.70353

Figure Lengend Snippet: Acetazolamide, tacrolimus, and LB‐100 impair the maintenance of KMT2A::AFF1+ B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). (A) Experimental design of the drug study to assess the effect of acetazolamide, tacrolimus, and LB‐100 in the maintenance of KMT2A::AFF1+ BCP‐ALL in NSG mice xenotransplanted with SEM cells. The treatment was initiated when 1% human cells were detected in the peripheral blood of NSG‐SEM mice. (B) Human cell contribution in the peripheral blood over the course of the 12‐day treatment. (C) Absolute number of SEM cells in the bone marrow of NSG mice (vehicle and drug) at Day 12. (D) Experimental design of the monotherapy drug study to assess the effect of acetazolamide on the survival of NSG mice xenotransplanted with cells derived from an infant KMT2A::AFF1+ BCP‐ALL patient. (E) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) of miR‐128a, miR‐130b, miR‐194, miR‐99b, and miR‐125a‐5p in leukemic cells from the infant KMT2A::AFF1+ BCP‐ALL patients used in (D). (F) Correlation of CA5B and miR‐194 expression in leukemic cell lines and the primary sample used in (D). (G) Human cell contribution in the peripheral blood 5 days after the end of the treatment. (H) Survival curve of NSG mice xenotransplanted with cells derived from an infant KMT2A::AFF1+ BCP‐ALL patient: vehicle‐ and acetazolamide‐treated. (I) Experimental design of combination therapy to assess the effect of acetazolamide alongside an induction therapy on the survival of NRG‐S PDX mice xenotransplanted with cells derived from an infant KMT2A::AFF1+ BCP‐ALL patient. (J) CA5B, PPP3CA, and PPP2R5C expression in an infant KMT2A::AFF1+ BCP‐ALL patient (Utrecht). (K) Total body luminescence signal (BLI) over time during and after the treatment for VXLC ( n = 6), VXLC + acetazolamide ( n = 6), and VXLC + acetazolamide + maintenance ( n = 5) groups. Data are presented as mean ± SEM and compared using a Welch's t ‐test. (L) Survival curve of VXLC‐, VXLC + acetazolamide‐, and VXLC + acetazolamide + maintenance‐treated groups. Survival differences were assessed using a Gehan–Breslow–Wilcoxon test. Data are presented as mean ± 95% CI. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Article Snippet: The primary antibody was added at a dilution of 1:1000 in 5% milk TBST Buffer and incubated overnight at 4°C, with mild shaking: human Carbonic Anhydrase VB/CA5B antibody (Biotechne Cat# AF3176), rabbit anti‐PPP2R5C antibody affinity purified (Bethyl Laboratories Inc Cat# A303‐814A), PPP3CA polyclonal antibody (Elabscience Cat# E‐AB‐14813), and monoclonal anti‐β‐actin antibody produced in mouse (Sigma Cat# A2228).

Techniques: Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing