P limk 1 2

Cells were lysed in standard lysis buffer (SLB; 10 mM K₂HPO₄, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.5% Triton X-100, 0.2% sodium deoxycholate; pH 7.4) supplemented with protease (cOmpleteTM, mini, EDTA-free; Roche, Mannheim, Germany; #1183617001) and phosphatase (PhosSTOP EASY pack; Roche, #04906837001) inhibitors, if not stated otherwise.
Western blotting was carried out as previously described [34 (link),36 (link),37 (link),83 (link),93 (link),94 (link)] using the following primary and secondary antibodies: Cofilin (1:2000, Cell Signaling, Frankfurt am Main, Germany; D3F9, #5175), p-Cofilin (Ser3, 1:1000, Cell Signaling, 77G2, #3313), Hsp60 (1:1000, Cell Signaling, D307, #4870), Pan-Cadherin (1:1000, Abcam, Cambridge, UK; ab6528), LIMK-1 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany; H-84, sc-5576), p-LIMK-1/2 (Thr508/505, 1:200, Santa Cruz, sc-28409-R), POD-F(ab’)₂-anti-Rabbit IgG (H+L, 1:5000, Jackson Immuno Research, Ely, UK; #711-036-152), and POD-anti-Mouse IgG (H+L, 1:5000, Jackson Immuno Research, #715-035-151). Signals were detected using Immobilon^® Western Chemiluminescent HRP Substrate (Merck, Darmstadt, Germany; #WBKLS0500) and an Odyssey^® Fc detection system (Li-Cor Biosciences, Bad Homburg vor der Höhe, Germany). Image processing and densitometric analysis was performed with the Li-Cor software Image Studio Lite (Ver. 5.2.5).

A total of 100 porcine oocytes were collected, lysed in Laemmli sample buffer (SDS sample buffer with 2-mercaptoethanol), and boiled at 100 °C for 10 min. Proteins were separated by SDS-PAGE and then electrophoretically transferred to polyvinylidene fluoride membranes. To avoid nonspecific binding, membranes were blocked with Tris-buffered saline (TBS) containing 0.1% (w/w) Tween 20 (TBST) and 5% (w/v) nonfat dry milk powder for 1 h at room temperature. The membranes were simultaneously incubated at overnight 4 °C with a rabbit polyclonal p-LIMK-1/2 (1:200, Santa Cruz, USA) or rabbit monoclonal anti-α-tubulin antibody (1:2,000; Cell Signaling Technology, Danvers, MA, USA). After washing three times in TBST (10 min each), the membranes were incubated for 2 h with secondary anti-rabbit HRP-conjugated antibodies (1:2,000; Cell Signaling Technology, Beverly, MA, USA) in 5% nonfat dry milk in TBST at room temperate. Finally, the membranes were washed 3 times in TBST and then the specific proteins were visualized using chemiluminescence reagent (Millipore, Billerica, MA).