primary antibodies specific for phosphorylated (p)-marcks (ser167/170; d13e4 xp)  (Cell Signaling Technology Inc)

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: ROS production is suppressed by PKCβ KO and PKC inhibitor Staurosporine, and MARCKS level and Ser167/170 phosphorylation are not affected in PKCβ KO cells. THP-1-derived PKCβ KO cells (generated using the CRISPR/Cas9 technique) and THP-1 WT cells were differentiated with 100 nM calcitriol for 5 d. Subsequently, ROS production was induced by PMA (100 nM) and determined (in RLU/s) via luminol-amplified chemiluminescence. ( A , B ) Kinetics ( A ) and the respective cumulated production (i.e., the AUC) within 60 min following stimulation ( B ) of total ROS generated by differentiated THP-1 WT and PKCβ KO cells (representative experiment, n = 3). ( C , D ) Kinetics ( C ) and the respective cumulated production within 60 min following stimulation ( D ) of intracellular ROS generated by differentiated THP-1 and PKCβ KO cells (representative experiment, n = 3). ( E ) Primary human monocytes were incubated in medium with Staurosprine (Stauro; 80 nM) for 2.5 h. Subsequently, PMA-induced total ROS production was assessed and depicted as cumulated ROS production within 30 min following stimulation (mean ± SD; n = 2). ( F ) THP-1 WT and PKCβ KO cells were treated with 100 nM PMA. At the indicated time points, cells were harvested. Total cell extracts were prepared, and MARCKS ED phosphorylation (Ser167/170) and protein levels were determined (Western Blot; loading control: GAPDH; representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Phospho-proteomics, Derivative Assay, Generated, CRISPR, Amplification, Incubation, Western Blot, Control

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: Increased monocytic ROS production following TNF long-term preincubation is dependent on MARCKS and PKCβ. ( A , B ) Kinetics ( A ) and the respective cumulated production (i.e., the AUC) within 180 min ( B ) of opsonized bacteria-induced (≥5 bacteria/cell) total ROS generated by 5 d calcitriol-differentiated THP-1 cells following preincubation with 80 ng/mL TNF for 48 h (i.e., added at day 3). Total ROS production was assessed via luminol-amplified chemiluminescence (in RLU/s) (representative experiment, n = 3). ( C , D ) Kinetics ( C ) and the respective cumulated production within 180 min ( D ) of opsonized bacteria-induced intracellular ROS generated by differentiated THP-1 cells following TNF preincubation as described in ( A ) (representative experiment, n = 3). ( E , F ) Kinetics ( E ) and the respective cumulated production within 30 min ( F ) of PMA-induced (100 nM) total ROS generated by primary human monocytes following TNF preincubation (80 ng/mL TNF for 48 h; representative experiment, n = 4). ( G ) Cumulated intracellular ROS production in MARCKS WT and KO clones (incubated as in ( A )) within 180 min following stimulation with bacteria (representative experiment, n = 3). ( H ) Cumulated intracellular ROS production in THP-1 and PKCβ KO cells (incubated as in ( A )) within 180 min following stimulation with bacteria (representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Bacteria, Generated, Amplification, Clone Assay, Incubation

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: Generation and characterization of MARCKS KO cells. Monocytic THP-1 cells were modified using the CRISPR/Cas9 technique to generate MARCKS KO cells. The resulting WT, IM, and KO clones were characterized in terms of allelic integrity, MARCKS protein expression, proliferation, monocytic differentiation, cytokine expression, and phagocytosis. ( A ) The table summarizes the condition of the 3 MARCKS alleles in MARCKS WT, IM, and KO clones as determined by Sanger sequencing. ( B ) MARCKS protein levels were detected in whole cell extracts of MARCKS WT, IM, and KO clones. Loading control: GAPDH (representative experiment, n > 3). ( C ) MARCKS WT and KO cells were synchronized for 3 d in minimal growth medium. Afterwards, cells were transferred into standard growth medium and cell numbers were counted daily (mean ± SD, n = 3). ( D ) Following calcitriol-induced differentiation (100 nM) for 3 d, CD14 expression on the cell surface was assessed using flow cytometry (dashed line: isotype control; representative experiment, n > 10). ( E ) Five-day calcitriol-differentiated MARCKS WT and KO cells were incubated with 80 ng/mL TNF for 2 h or 48 h (TNF added at day 3), and the IL-8 mRNA level was determined by qRT-PCR. The IL-8 level in differentiated control cells (i.e., in the absence of TNF) was set as 1 (mean ± SD, representative experiment determined in triplicates; n = 3). ( F ) Five-day differentiated THP-1 as well as MARCKS WT and KO cells were incubated with opsonized eFlour670-labeled bacteria for 1 h (>5 bacteria per cell; mean ± SD, representative experiment, determined in duplicates; n = 4). During the incubation with bacteria, control cells were kept on ice. Phagocytosis was analyzed using flow cytometry. The phagocytic activity (i.e., the geometric mean fluorescence intensity) of living and bacteria-positive cells was calculated, and the value obtained in differentiated MARCKS WT cells was set as 100% (dashed line).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Modification, CRISPR, Clone Assay, Expressing, Sequencing, Control, Flow Cytometry, Incubation, Quantitative RT-PCR, Labeling, Bacteria, Activity Assay, Fluorescence

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: PMA-induced ROS production is suppressed in monocytic MARCKS KO cells. ( A – D ) Monocytic THP-1-derived MARCKS WT and KO clones were differentiated with 100 nM calcitriol for 5 d. ROS production was induced by 100 nM PMA and assessed via luminol-amplified chemiluminescence in relative light units per second (RLU/s). ( A , B ) PMA-induced total ROS production is completely abolished in monocytic MARCKS KO cells. Kinetics ( A ) and the respective cumulated total ROS production (i.e., the area under the curve (AUC) within 60 min following PMA stimulation) ( B ) of total ROS production in MARCKS WT and KO clones (representative experiment, n = 3). ( C , D ) PMA-induced intracellular ROS production is strongly inhibited in MARCKS KO and partially reduced in MARCKS IM cells. Kinetics (( C ), representative experiment) and the respective cumulated intracellular ROS production within 60 min following PMA stimulation (( D ), mean ± SD, n = 3) in WT, IM, and KO clones. ( E ) Reconstitution of MARCKS in KO cells restores PMA-induced ROS production. MARCKS KO clones were differentiated with 100 nM calcitriol for 5 d. At day 3, cells were transfected with a MARCKS-expressing vector or the respective vector control (control cells: THP-1, transfection control: EGFP-encoding plasmid). Cumulated ROS production of EGFP-positive (i.e., transfected) cells within 60 min following PMA stimulation was detected by flow cytometry. The ROS production in control-transfected KO cells was set as 1 (mean ± SD, n = 3). The insets show the MARCKS levels in KO and THP-1 cells transfected with the control or the MARCKS expression vector. ( F ) Basal Akt levels are reduced and Akt re-phosphorylation is delayed in MARCKS KO cells. THP-1 WT and MARCKS KO cells were treated with 100 nM PMA. At the indicated time points, cells were harvested, and total cell extracts were prepared. Akt phosphorylation (at Thr308) and total Akt levels were determined via Western Blot (loading control: GAPDH; representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Derivative Assay, Clone Assay, Amplification, Transfection, Expressing, Plasmid Preparation, Control, Flow Cytometry, Phospho-proteomics, Western Blot

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: Bacteria-induced total ROS production is markedly inhibited, and intracellular ROS production is completely abolished in MARCKS KO cells. MARCKS WT and KO cells were differentiated with 100 nM calcitriol for 5 d. ROS production induced by opsonized bacteria (bac; >5 bacteria/cell) was assessed via luminol-amplified chemiluminescence in RLU/s. ( A , B ) Kinetics ( A ) and the respective cumulated total ROS production within 180 min ( B ) in MARCKS WT and KO clones following stimulation (representative experiment, n = 4). ( C , D ) Kinetics ( C ) and the respective cumulated intracellular ROS production within 180 min ( D ) in WT and KO clones following stimulation (representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Bacteria, Amplification, Clone Assay

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: ROS production is suppressed by PKCβ KO and PKC inhibitor Staurosporine, and MARCKS level and Ser167/170 phosphorylation are not affected in PKCβ KO cells. THP-1-derived PKCβ KO cells (generated using the CRISPR/Cas9 technique) and THP-1 WT cells were differentiated with 100 nM calcitriol for 5 d. Subsequently, ROS production was induced by PMA (100 nM) and determined (in RLU/s) via luminol-amplified chemiluminescence. ( A , B ) Kinetics ( A ) and the respective cumulated production (i.e., the AUC) within 60 min following stimulation ( B ) of total ROS generated by differentiated THP-1 WT and PKCβ KO cells (representative experiment, n = 3). ( C , D ) Kinetics ( C ) and the respective cumulated production within 60 min following stimulation ( D ) of intracellular ROS generated by differentiated THP-1 and PKCβ KO cells (representative experiment, n = 3). ( E ) Primary human monocytes were incubated in medium with Staurosprine (Stauro; 80 nM) for 2.5 h. Subsequently, PMA-induced total ROS production was assessed and depicted as cumulated ROS production within 30 min following stimulation (mean ± SD; n = 2). ( F ) THP-1 WT and PKCβ KO cells were treated with 100 nM PMA. At the indicated time points, cells were harvested. Total cell extracts were prepared, and MARCKS ED phosphorylation (Ser167/170) and protein levels were determined (Western Blot; loading control: GAPDH; representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Phospho-proteomics, Derivative Assay, Generated, CRISPR, Amplification, Incubation, Western Blot, Control

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: Increased monocytic ROS production following TNF long-term preincubation is dependent on MARCKS and PKCβ. ( A , B ) Kinetics ( A ) and the respective cumulated production (i.e., the AUC) within 180 min ( B ) of opsonized bacteria-induced (≥5 bacteria/cell) total ROS generated by 5 d calcitriol-differentiated THP-1 cells following preincubation with 80 ng/mL TNF for 48 h (i.e., added at day 3). Total ROS production was assessed via luminol-amplified chemiluminescence (in RLU/s) (representative experiment, n = 3). ( C , D ) Kinetics ( C ) and the respective cumulated production within 180 min ( D ) of opsonized bacteria-induced intracellular ROS generated by differentiated THP-1 cells following TNF preincubation as described in ( A ) (representative experiment, n = 3). ( E , F ) Kinetics ( E ) and the respective cumulated production within 30 min ( F ) of PMA-induced (100 nM) total ROS generated by primary human monocytes following TNF preincubation (80 ng/mL TNF for 48 h; representative experiment, n = 4). ( G ) Cumulated intracellular ROS production in MARCKS WT and KO clones (incubated as in ( A )) within 180 min following stimulation with bacteria (representative experiment, n = 3). ( H ) Cumulated intracellular ROS production in THP-1 and PKCβ KO cells (incubated as in ( A )) within 180 min following stimulation with bacteria (representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Bacteria, Generated, Amplification, Clone Assay, Incubation