triton x 100 rabbit anti mct2  (Alomone Labs)

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Lactate metabolism is required for inner retinal neuron survival. A , immunofluorescence labeling of retinal transverse cryosections showing MCT2 expression mainly in the inner nuclear layer (INL). Colabeling for PKCα and calretinin illustrates MCT2 expression in RBCs and a subset of ACs. B i – B iv ( top ), ( C ) statistical analysis, and quantification of the cell death assay (TUNEL), performed in organotypic retinal explants, showing occasional TUNEL positive nuclei ( arrows ). When compared to control (CTR, n = 5) treatment with AR-C and Shikonin increased INL cell death, while SR treatment did not. The data were analyzed with the Kruskal-Wallis and Dunn’s multiple comparison post hoc tests. B i -B iv ( bottom ), ( D ) quantification of RBCs per 100 μm retinal length revealed a significant density reduction of this cell type after treatment with SR, AR-C, and Shikonin, supporting a dependence of RBCs on extracellular lactate. The data were analyzed by one-way ANOVA with Tukey’s multiple comparison post hoc test. Each dot reflects a single retinal explant. E , schematic summary, showing the transporters, applied drugs used throughout the investigation, and their respective effects on lactate metabolism. Shikonin and FX-11 inhibit lactate synthesis directly, while AR-C155858 and SR-13800 block lactate transport. Graphs display mean values ± SD; asterisks indicate ∗ p < 0.05, ∗∗ p < 0.01. SR = MCT1 inhibitor; Shikonin = PKM2 inhibitor. ACs, amacrine cells; AR-C = MCT2 inhibitor; INL, inner nuclear layer; LDH-A, lactate dehydrogenase A; MCT1, monocarboxylate transporter one; MCT2, monocarboxylate transporter 2; ONL, outer nuclear layer; PKCα, protein kinase Cα; PKM2, pyruvate kinase M2; RBCs, rod bipolar cells.

Article Snippet: The primary antibodies, anti-MCT2 (SLC16A7) (Alomone labs, Cat. No. AMT-012, RRID: AB_2340997 ), Anti-protein kinase Cα (Thermo Fisher Scientific, Cat. No. MA1-157, RRID: AB_2536865 ), and anti-calretinin (Abcam, Cat. No. A85366, RRID: AB_2748943 ) were diluted one:100 in blocking solution and incubated at 4 °C overnight.

Techniques: Immunofluorescence, Labeling, Expressing, TUNEL Assay, Control, Comparison, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Depolarization of inner retinal cells is reduced and slowed by inhibition of lactate metabolism. A and D , representative images of Fluo4-AM and CoroNa Green-loaded cells in the INL, before and at two time points after bath perfusion with KCl, in controls and retinas incubated with SR, AR-C, and Shikonin. B and E , ( top ), traces of individual experiments in each condition ( red line = mean, gray shadow = SD). B and E , ( bottom ), overview of the kinetic parameters measured in the imaging. C i–iv , statistical analysis of the different parameters in the calcium imaging experiments indicating alterations in the amplitude and time to peak. F i–iv , statistical analysis of the sodium imaging experiments showing specific alterations in all the parameters after MCT2 inhibition. Box plots display the median ± min and max values and the mean in red . Individual values are displayed as open circles ( gray ). The control is represented as a dashed line at 100%, and results are presented as percentage of control. Asterisks indicate ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. INL, inner nuclear layer; MCT, SR= MCT1 inhibitor; AR-C= MCT2 inhibitor; Shikonin = PKM2 inhibitor; FX-11= LDH-A inhibitor. See also Figs. S1 and . LDH-A, lactate dehydrogenase A; MCT, monocarboxylate transporter.

Article Snippet: The primary antibodies, anti-MCT2 (SLC16A7) (Alomone labs, Cat. No. AMT-012, RRID: AB_2340997 ), Anti-protein kinase Cα (Thermo Fisher Scientific, Cat. No. MA1-157, RRID: AB_2536865 ), and anti-calretinin (Abcam, Cat. No. A85366, RRID: AB_2748943 ) were diluted one:100 in blocking solution and incubated at 4 °C overnight.

Techniques: Inhibition, Incubation, Imaging, Control

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Combined MCT1/MCT2 inhibition decreases intracellular lactate levels in inner retinal neurons. A – C , ( left ), from left to right : fluorescence curves showing lactate influx evoked by stimulation with 10 mM lactate, a diagram showing the transporters affected by the application of each drug and a fluorescence trace showing the effect of each drug on the lactate influx. A – C , ( right ), statistical analysis of the response amplitude. An evident response disruption was noted only when MCT2 was inhibited. However, under MCT1 and MCT4 inhibition, the amplitude was unaltered. D , modulation of intracellular lactate levels by transient depolarization. E , ( top ), effects of the inhibition of different MCTs and retinal depolarization on lactate dynamics. Black dashed lines indicate drug-induced change in slope and red dashed lines indicate the effect of drug + KCl. E , ( bottom ), statistical analysis of the slope of the responses under basal conditions (only drug, left ) and depolarization (drug + K + , right ). The data revealed that MCT2 inhibition led to a reduction in intracellular lactate levels, resulting from lactate consumption. This consumption was exacerbated after depolarization in SR, AR-C, and Syro. Data were analyzed using either a paired Student’s t test or Wilcoxon matched-pair test. The black trace represents the average of one experiment, whereas the light gray shadow represents the SD. Graphs display the mean ± SD. ∗ Indicates p < 0.05. SR = MCT1 inhibitor; AR-C = MCT1 and MCT2 inhibitor; Syro = MCT1 and MCT4 inhibitor. The number of experiments is represented as N = number of explants and n = number of cells recorded. See also Figs. S5 and . MCT, monocarboxylate transporter.

Article Snippet: The primary antibodies, anti-MCT2 (SLC16A7) (Alomone labs, Cat. No. AMT-012, RRID: AB_2340997 ), Anti-protein kinase Cα (Thermo Fisher Scientific, Cat. No. MA1-157, RRID: AB_2536865 ), and anti-calretinin (Abcam, Cat. No. A85366, RRID: AB_2748943 ) were diluted one:100 in blocking solution and incubated at 4 °C overnight.

Techniques: Inhibition, Fluorescence, Disruption

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Inhibition of lactate transport through MCT2 induces alterations in RBC currents. A and C , comparison of the voltage-current relationship of the outward current and calcium current under different conditions. B and D , representative recordings to depolarizing voltage steps. Reciprocal feedback was altered only in the AR-C condition ( p = 0.0042) but was unaffected in the SR condition ( p = 0.1957) compared to controls. E and F , in the presence of 20 mM lactate, we observed a decrease in the outward current ( p = 0.0224) and calcium current ( p = 0.0003) only under MCT2 inhibition, but no differences were noted either in the outward current ( p = 0.3925) or calcium current ( p = 0.8146) when only MCT1 was blocked. G , similar results were obtained when we measured the membrane potential, where AR-C ( p = 0.0321) caused a significant depolarization, while the SR condition was not different from controls ( p = 0.0822). Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc test. Each dot represents a single recorded cell. Graphs represent the mean ± SD; asterisks indicate ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. SR = MCT1 inhibitor; AR-C= MCT1 and MCT2 inhibitor. See also Fig. S3 and , and Table 1 . MCT, monocarboxylate transporter; RBC, rod bipolar cell.

Article Snippet: The primary antibodies, anti-MCT2 (SLC16A7) (Alomone labs, Cat. No. AMT-012, RRID: AB_2340997 ), Anti-protein kinase Cα (Thermo Fisher Scientific, Cat. No. MA1-157, RRID: AB_2536865 ), and anti-calretinin (Abcam, Cat. No. A85366, RRID: AB_2748943 ) were diluted one:100 in blocking solution and incubated at 4 °C overnight.

Techniques: Inhibition, Comparison, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Lactate dynamics of MCs under different conditions. A and B , effect of transient depolarization on intracellular glucose ( A ) and lactate ( B ) levels. C , lactate dynamics after inhibition of different MCTs and retinal depolarization. Black dashed lines indicate drug-induced changes in slope and red dashed lines indicate the effect of drugs + KCl. D , statistical analysis of the slope of the responses in the basal condition (drug only) and under depolarization (drug + K+). Accumulation of intracellular lactate was observed after bath application of different MCT inhibitors. However, this increase was less intense after depolarization under all conditions. Data were analyzed either with paired Student’s t test or Wilcoxon matched-pairs test. Graphs display the mean ± SD. ∗ Indicates p < 0.05. SR = MCT1 inhibitor; AR-C = MCT1 and MCT2 inhibitor; Syro = MCT1 and MCT4 inhibitor. The black trace represents the average response, while the light gray shadow represents the SD. The number of experiments is represented as: N = number of explants; n = number of cells recorded. MCs, Müller cells; MCT, monocarboxylate transporter.

Article Snippet: The primary antibodies, anti-MCT2 (SLC16A7) (Alomone labs, Cat. No. AMT-012, RRID: AB_2340997 ), Anti-protein kinase Cα (Thermo Fisher Scientific, Cat. No. MA1-157, RRID: AB_2536865 ), and anti-calretinin (Abcam, Cat. No. A85366, RRID: AB_2748943 ) were diluted one:100 in blocking solution and incubated at 4 °C overnight.

Techniques: Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Model for th e lactate dy namics in inner retinal cells. General model proposing a consumption of lactate by inner retinal cells. Previous work demonstrated the functional expression of MCT1, MCT2, and MCT4 in MCs, where MCT2 (and to a minor degree also MCT1) mainly regulates lactate influx, while MCT4 mediates lactate efflux, contributing to the accumulation of extracellular lactate. Here, we propose that this extracellular lactate produced by MCs and possibly other retinal cell types is consumed by BCs and ACs through MCT2. ACs, amacrine cells; BCs, bipolar cells; MCs, Müller cells; MCT1, monocarboxylate transporter one; MCT2, monocarboxylate transporter 2; MCT4, monocarboxylate transporter 4.

Article Snippet: The primary antibodies, anti-MCT2 (SLC16A7) (Alomone labs, Cat. No. AMT-012, RRID: AB_2340997 ), Anti-protein kinase Cα (Thermo Fisher Scientific, Cat. No. MA1-157, RRID: AB_2536865 ), and anti-calretinin (Abcam, Cat. No. A85366, RRID: AB_2748943 ) were diluted one:100 in blocking solution and incubated at 4 °C overnight.

Techniques: Functional Assay, Expressing, Produced