Journal: Cells

Article Title: Phosphorylation-Dependent Differences in CXCR4-LASP1-AKT1 Interaction between Breast Cancer and Chronic Myeloid Leukemia

doi: 10.3390/cells9020444

Figure Lengend Snippet: LASP1 regulates AKT1-S473 phosphorylation. ( A ) Representative Western blots analyzing LASP1, pERK, AKT1, pAKT1-T308 and pAKT1-S473 after LASP1 knockdown in MDAMB-231 (stably transfected with shRNA-LASP1 or nonsense shRNA and downregulated by inducible doxycyclin treatment (depicted with (+) and (−))), in MCF-7 breast cancer cells (transiently transfected with different LASP1 specific siRNA), and in LASP1-knockout K562 single cell lines. ( B ) Impaired AKT1-S473 phosphorylation after LASP1 depletion. Statistical differences were analyzed using the paired t-test ( n = 6).

Article Snippet: Equal amounts of cells (1 × 10 4 ) were analyzed by immunoblotting with the following antibodies: LASP1 [ ] (diluted 1:2000); anti-β-Actin (1:3000, #1616, Santa Cruz, Dallas, CA, USA); pLASP1-S146 (1:700, IgG-1445, Immunoglobe, Himmelstadt, Germany); pLASP1-Y171 (1:500, IgG-1418, Immunoglobe); AKT1 (1:100, SAB 4500802, Sigma-Aldrich, Darmstadt, Germany); pAKT-S473 (1:1000, #9271, Cell Signaling, Danvers, MA, USA); pAKT-T308 (1:1000, #9275, Cell Signaling), pERK 1:500, #9102, Cell Signaling), HA-tag (1:700, sc-7392, Santa Cruz, Dallas, TX, USA).

Techniques: Phospho-proteomics, Western Blot, Knockdown, Stable Transfection, Transfection, shRNA, Knock-Out

Journal: Cells

Article Title: Phosphorylation-Dependent Differences in CXCR4-LASP1-AKT1 Interaction between Breast Cancer and Chronic Myeloid Leukemia

doi: 10.3390/cells9020444

Figure Lengend Snippet: Role of CXCR4 stimulation in LASP1 phosphorylation in CXCR4 expressing HEK-293 cells. ( A ) Representative Western blot analysis of LASP1, pLASP1-Y171, pLASP1-S146, AKT1, and pAKT-S473 in HEK-293 cells transfected with CXCR4 and stimulation with the CXCR4 agonist CXCL12 for the time points indicated. β-Actin served as loading control. ( B ) Densitometric quantification of phosphorylation. Data revealed a two-fold increase in pAKT1-S473 and a two-fold increase in pLASP-Y171 phosphorylation concomitant with a 50% decrease in LASP1-S146 phosphorylation in CXCR4 expressing HEK-293 cells compared to CXCR4-deficient HEK-293 cells after 2 min CXCL12 stimulation, which declined to basal levels after 10 min. Comparable results were obtained in two independent experiments.

Article Snippet: Equal amounts of cells (1 × 10 4 ) were analyzed by immunoblotting with the following antibodies: LASP1 [ ] (diluted 1:2000); anti-β-Actin (1:3000, #1616, Santa Cruz, Dallas, CA, USA); pLASP1-S146 (1:700, IgG-1445, Immunoglobe, Himmelstadt, Germany); pLASP1-Y171 (1:500, IgG-1418, Immunoglobe); AKT1 (1:100, SAB 4500802, Sigma-Aldrich, Darmstadt, Germany); pAKT-S473 (1:1000, #9271, Cell Signaling, Danvers, MA, USA); pAKT-T308 (1:1000, #9275, Cell Signaling), pERK 1:500, #9102, Cell Signaling), HA-tag (1:700, sc-7392, Santa Cruz, Dallas, TX, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Transfection, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Cetuximab–siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib

doi: 10.1177/15330338211041453

Figure Lengend Snippet: CRISPR–Cas9 knockout analysis of KRAS in NCI-H23 cells. (A) Western blot analysis after knockout of KRAS mutation. Almost-complete knockout is observed for KRAS protein, while downregulation of pMEK and pAKT is observed. ** P < 0.01. (B) RFP expression in transfected cells indicates successful transfection and gene modification.

Article Snippet: Protein Assay Dye Reagent Concentrate was purchased from bio-rad (Cat # 5000002). β-Actin (Cat # 8457S), rabbit monoclonal pMEK 1/2 (S221, Cat # 9154S), rabbit monoclonal MEK 1/2 (D1A5, Cat # 8727S), rabbit monoclonal AKT (Cat # 4691S), and rabbit monoclonal pAKT (T308, Cat # 4060S) were purchased from Cell Signalling Technologies.

Techniques: CRISPR, Knock-Out, Western Blot, Mutagenesis, Expressing, Transfection, Modification