Journal: International Journal of Molecular Sciences

Article Title: Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

doi: 10.3390/ijms17101696

Figure Lengend Snippet: Effects of NaB on the interaction between MLCK and calmodulin as well as phosphorylation levels of MLC2 in Caco-2 cell monolayers. ( A ) After Ca 2+ switch, Caco-2 cells were cultured in normal Caco-2 medium with or without 2 mmol/L of NaB. Co-Immunoprecipitation (Co-IP) of MLCK and calmodulin was performed at 0, 4 or 8 h, respectively; ( B ) The quantification of MLCK immunoreactive signals by normalized to calmodulin signals in ( A ); ( C ) The change of TERs after Ca 2+ switch under the condition of 2 mmol/L of NaB, or 250 μmol/L of Permeant inhibitor of MLC kinase (PIK) at 0, 2, 4 and 8 h, respectively; ( D ) Total cell lysates from untreated cells or those treated with 2 mmol/L of NaB or 250 μmol/L of PIK were subjected to immunoblotting for pSer19-MLC2, total MLC2 and GAPDH, respectively; ( E ) MLC2 activity was expressed as the ratio of the phosphorylated form of the MLC2 to total MLC2. Values are means ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and controls as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference ( p < 0.05) between NaB and NaB/PIK. PIK-Permeant inhibitor of MLC kinase.

Article Snippet: The target proteins on membranes were respectively blotted using primary antibodies of S19 phospho-MLC2 (1:2000), MLC2 (1:1000), (Cell Signaling Technology, Danvers, MA, USA), PKCβ1 (1:500), PKCβ2 (phosphor S660) (1:50,000, Abcam, USA), p-PKCβ1 (Thr641) (1:200), PKCβ2 (1:200, Santa Cruz, CA, USA), and GAPDH (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Phospho-proteomics, Cell Culture, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

doi: 10.3390/ijms17101696

Figure Lengend Snippet: The phosphorylation change of MLC2 and PKCβ2 during TJs assembly induced by NaB. ( A ) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 10 umol/L of SKF-96365; ( B , C ) The ratio of phosphorylated MLC2 to MLC2 or phosphorylated PKCβ2 to PKCβ2 in ( A ); ( D ) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 5 μmol/L of LY-333531; ( E ) The ratio of phosphorylated MLC2 to MLC2 in ( D ); ( F ) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 10 μmol/L of Compound C; ( G ) The ratio of phosphorylated MLC2 to MLC2 in ( F ). All experiments were performed at 8 h after calcium switch. Data represent mean ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and control group as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference between indicated groups, p < 0.05. CC—Compound C.

Article Snippet: The target proteins on membranes were respectively blotted using primary antibodies of S19 phospho-MLC2 (1:2000), MLC2 (1:1000), (Cell Signaling Technology, Danvers, MA, USA), PKCβ1 (1:500), PKCβ2 (phosphor S660) (1:50,000, Abcam, USA), p-PKCβ1 (Thr641) (1:200), PKCβ2 (1:200, Santa Cruz, CA, USA), and GAPDH (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

doi: 10.3390/ijms17101696

Figure Lengend Snippet: Proposed diagram showing the mechanism of NaB on reassembly of tight junctions in Caco-2 monolayers. NaB appears to activate Store-Operated Ca 2+ Channel (SOCC) which conducts the Ca 2+ influx and then activates CaMKKβ/AMPK, resulting in PKCβ2 and MLCK/MLC2 pathways to mediate barrier function recovery. The black arrows indicate active effects, and the line with bar at the end indicates inhibition effect. The red arrow indicates reduction, and the green arrows indicates increase.

Article Snippet: The target proteins on membranes were respectively blotted using primary antibodies of S19 phospho-MLC2 (1:2000), MLC2 (1:1000), (Cell Signaling Technology, Danvers, MA, USA), PKCβ1 (1:500), PKCβ2 (phosphor S660) (1:50,000, Abcam, USA), p-PKCβ1 (Thr641) (1:200), PKCβ2 (1:200, Santa Cruz, CA, USA), and GAPDH (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Inhibition