Figure 3.

Hyperosmotically induced (0.8 M sorbitol for 30 min) phosphorylation of alpha-tubulin and HvMPK4 as probed in barley root extracts. (A1) Severe, non-ionic hyperosmotic stress induces activation of unknown MAPKs recognized by pTEpY antibody. (A2) Under the same conditions total levels of alpha-tubulin are slightly upregulated as evidenced in immunoblots following standard SDS-PAGE. (B1) Phos-Tag™ gel separates alpha-tubulin to a phosphorylated (top arrow, pTUA) and a non-phosphorylated band (bottom arrow TUA) in the presence of Mn²⁺. (B2) A wider separation of phosphorylated (top arrow, pTUA) from non-phosphorylated alpha tubulin (bottom arrow, TUA) is seen in the presence of Zn²⁺ in the Phos-Tag™ gel indicating better efficiency of Zn²⁺ containing Phos-Tag™ gels to separate phosphorylated tubulin. The panels on the left of (A1) corresponds to image of the protein ladder following Ponceau S staining of the membrane. (C1) Identical extracts as those used in Panel (A) where probed with anti-HvMPK4 antibody. The antibody specifically recognizes HvMPK4 (left arrow) and further shows conservation of total HvMPK4 levels in control and hyperosmotically-treated samples. (C2) Hyperosmotically-induced phosphorylation of HvMPK4 as monitored by Zn²⁺/Phos-Tag™ gel electrophoresis and western blot. The lower band corresponds to unphosphorylated HvMPK4 (bottom arrow) while the upper bands correspond to different phosphorylation statuses of HvMPK4 after hyperosmotic induction (right lane, top arrows, pHvMPK4). In untreated samples (left lane) only one faint upper band appears probably corresponding to basally phosphorylated HvMPK4. Panels on the left of (A1) and (C1) correspond to images of the protein ladder following Ponceau S staining of the membranes.